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WO1992007940A2 - Bovine respiratory syncytial virus genes - Google Patents

Bovine respiratory syncytial virus genes Download PDF

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Publication number
WO1992007940A2
WO1992007940A2 PCT/US1991/008177 US9108177W WO9207940A2 WO 1992007940 A2 WO1992007940 A2 WO 1992007940A2 US 9108177 W US9108177 W US 9108177W WO 9207940 A2 WO9207940 A2 WO 9207940A2
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brsv
sequence
protein
leu
ser
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PCT/US1991/008177
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WO1992007940A3 (en
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Siba Kumar Samal
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Siba Kumar Samal
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Bovine Respiratory Syncytial Virus is an RNA virus which is recognized as an important cause of lower respiratory disease of cattle in Asia, Europe and the United States. In cattle, BRSV infection is more significantly associated with respiratory disease than any other virus. Furthermore, the highest incidence of severe BRSV disease in cattle is between 2 and 4.5 months of age. About 70 percent of cattle have been infected by BRSV by 9 months of age.
  • BRSV infection The characteristics of BRSV infection include extensive damage to the mucuous membranes, leaving the respiratory tract susceptible to dust, debris and secondary infectious agents. BRSV infection also causes a condition which resembles systemic anaphylaxis or hypersensitivity. Outbreaks of BRSV infection usually last about 10 to 14 days, and are characterized by high mortality.
  • BRSV is not easily grown, is closely associated with the cell membrane and is inherently unstable (Stott and Taylor, Arch. Virolo ⁇ v 84. 1 (1985)). Therefore, purification of the virus by biophysical techniques is extremely difficult.
  • BRSV human respiratory syncytial virus
  • HRSV human respiratory syncytial virus
  • pneumonia virus of mice comprise the genus Pneumovirus which is within the family Paramyxoviridae.
  • Viruses of this family have enveloped pleomorphic virions which contain helical, elongated nucleocapsids.
  • the genome is linear, single-stranded RNA which replicates in the cytoplasm.
  • the BRSV virion appears as round or pleomorphic forms which measure about 80 to 500 mm across, or as filamentous forms up to several um in length.
  • the outer membrane of the virion is studded with projections about 12 mm long, each of which is about 10 mm apart.
  • Purified virions contain a unique species of single-stranded RNA of which at least 93 percent is negative sense. Discrepancies regarding the size and number of polypeptides in the virion are due, in part, to differences between virus strains (Stott and Taylor, supra) .
  • the smaller polypeptide has a molecular weight between about 10,000 and 13,000 daltons, and the. larger polypeptide has a molecular weight between about 19,000 and 25,000 daltons.
  • the large polypeptide is a non-glycosylated protein. Neither of the small polypeptides has any known functions (Stott and Taylor, supra) .
  • M protein A larger protein, the M protein (about 27,000 to 28,000 daltons) is believed to be the membrane protein.
  • the M protein has 256 amino acids, and is relatively basic with two hydrophobic regions in the C-terminal third of the protein.
  • the P protein is a phosphorylated protein with a molecular weight of about 32,000 to 38,000 daltons. This protein is associated with the nucleocapsid.
  • nucleocapsids isolated from purified BRSV contain primarily NP protein in association with RNA.
  • the NP protein has a molecular weight of between about 40,000 and 44,000 daltons, and has 467 amino acids, most of which are basic amino acids.
  • F protein Two glycoproteins which are believed to be located on the surface of the virion are the F protein and G protein.
  • the F protein has a molecular weight of between about 66,000 and 68,000 daltons
  • the G protein has a molecular weight of between about 79,000 and 90,000 daltons. These two proteins have a rod-shaped morphology, suggesting that they may be the studded projections of the virion.
  • the F protein is comprised of two smaller glycoproteins linked by disulfide bonds.
  • One of the smaller glycoprotein has a molecular weight between about 43,000 and 56,000 daltons and the other smaller glycoprotein has a molecular weight between about 19,000 and 22,000 daltons.
  • the F protein has been shown to be the fusion protein by the inhibition of cell fusion by a monoclonal antibody to the F protein.
  • the G protein is believed to be the attachment protein of the virion.
  • Monoclonal antibodies to either the F protein or the G protein neutralize infectivity of the virus.
  • L protein There is little known about the largest polypeptide, the L protein. This protein has a molecular weight of between about 160,000 daltons and 200,000 daltons and is believed to be the RNA polymerase of the virion.
  • BRSV and HRSV differ in plaque reduction tests using bovine sera, cattle have been reported to be equally protected from BRSV infection by either BRSV or HRSV antibodies. Any differences detected by neutralization are believed to reflect changes in the epitopes on either the F protein or G protein (Stott and Taylor, supra) . Furthermore, two monoclonal antibodies to the BRSV fusion protein appear to react complement fixation and immunofluoresence tests, all strains of respiratory syncytial virus cross-react (Stott and Taylor, supra) .
  • the three vaccines which have been tested are: 1) an inactivated antigen combined with adjuvant given intramuscularly; 2) live attenuated viruses given intranasally; and 3) live modified virus given intramuscularl .
  • BRSV and HRSV have traditionally been classified in the same genus, Pneumovirus. as stated above, at least one group of researchers have suggested that these two viruses be classified in separate groups of the genus (Lerch et al., J. Virol. 64, 5559 (1990)).
  • a comparison of the amino acid sequences of the G protein of BRSV with the G protein of either subgroup A or B of HRSV showed only a 29 to 30% amino acid identity.
  • antisera to the BRSV G protein made by using a recombinant vector to immunize animals, recognized the BRSV G protein but not the HRSV G protein, and vice versa (Lerch et al. , supra) .
  • the present invention relates to genes derived from BRSV, vectors produced with the genes and expression systems for the genes.
  • the genes comprise the nucleotide sequences set forth as in the Sequence Listing as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6; SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14; SEQ ID NO:16, and SEQ ID NO:18.
  • the invention further relates to fragments of these genes.
  • the invention also encompasses diagnostic probes comprising these genes or fragments thereof. All of the genes or their fragments are useful as diagnostic probes.
  • the invention further encompasses purified BRSV proteins or fragments thereof. These purified proteins and fragments can be used to detect BRSV antibodies, and can be used as vaccines.
  • the purified proteins which are useful for the detection of BRSV antibodies and as vaccines are proteins set forth in the Sequence Listing as SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19.
  • the protein of SEQ ID NO:3 is particularly useful for diagnostic testing for the presence of BRSV antibodies.
  • a further aspect of the invention are antibodies to the gene products or fragments thereof.
  • Fig. 1 shows the genetic map of BRSV strain A2
  • Fig. 2 shows an SDS-PAGE gel in which [ 3 H] glucosamine labeled antigens were immunoprecipitated with BRSV (strain A51908) antiserum; and
  • Fig. 3 shows an autoradiograph of the hybridization of 3 P-labeled BRSV-N gene probe to the RNA of a variety of bovine respiratory viruses.
  • Adjacent A position in a nucleotide sequence immediately 5' or 3' to a defined sequence.
  • Cell Culture A proliferating mass of cells which may be in an undifferentiated or differentiated state.
  • “cell”, “cell line”, and “cell culture” are used interchangeably and all such designations include progeny.
  • progeny includes the primary subject cell and cultures derived therefrom without regard for the number of tranfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny which have the same functionality as sreened for in the originally transformed cell, are included. Where distinct designations are intended, it will be clear from the context.
  • Coding Sequence A deoxyribonucleotide sequence which when transcribed and translated results in the formation of a cellular protein, or a ribonucleotide sequence which when translated results in the formation of a cellular protein.
  • Control Sequences refers to DNA sequences necessary for the expression of an operablylinked coding sequence in a particular host organism.
  • the control sequences which are suitable for procaryotes include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood, sequences.
  • Eucaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Expression System Refers to DNA sequences containing a desired coding sequence and control sequences in operable linkage, so that hosts transformed with these sequences are capable of producing the encoded proteins.
  • the expression system may be included on a vector; however, the relevant DNA may then also be integrated into the host chromosome.
  • Gene A discrete nucleic acid region which is responsible for a discrete cellular product.
  • Operably Linked refers to juxtaposition such that the normal function of the components can be performed.
  • a coding sequence "operably linked" to control equences refers to a configuratin wherein the coding sequence cn be expressed under the control of these sequences.
  • Promoter The 5'- flanking, non-coding sequence adjacent a coding sequence which is involved in the initiation of transcription of the coding sequence.
  • Substantial Sequence Homology Donates nucleotide sequences that are substantially functionally equivalent to one another. Nucleotide differences between such sequences having substantial sequence homology will be de minimus in affecting the function of the gene products or an RNA coded for such a sequence.
  • the present invention relates to the novel genes and gene fragments derived from BRSV.
  • the entire genome for BRSV strain A 51908 is presented in SEQ ID NO:l.
  • the genes and gene fragments thereof are:
  • nucleocapsid protein gene (the N gene) (SEQ ID NO:2) ;
  • the matrix protein gene (the M gene) (SEQ ID NO:4) ;
  • the phosphoprotein gene (the P gene) (SEQ ID NO:6) ;
  • the fusion protein gene (the F gene) (SEQ ID NO:10) ;
  • glycoprotein gene (the G gene) (SEQ ID NO:12) ;
  • genes were derived from two strains of BRSV.
  • the genes designated by SEQ ID NOS. 2, 4, 6, 8, 10, 12 and 14 above were derived from BRSV strain A 51908, which is available from the American Type * Culture Collection (ATCC #VR-794) .
  • the other strain is strain FS-l, which is available from the National Veterinary Services Laboratory (NVSL) U.S.D.A., Ames, Iowa.
  • NVSL National Veterinary Services Laboratory
  • the P gene of BRSV strain FS-l is set forth in the Sequence Listing as SEQ ID No:16, and the F gene of this strain is set forth as SEQ ID NO:18.
  • the present inventors have compared the polypeptides of four strains of BRSV. Based on the size of the F 2 fragment and P protein the BRSV strains examined could be classified into groups.
  • Fig. 2 shows the different migration patterns of the F 2 protein of different strains of BRSV.
  • lanes 3-6 show four different strains of BRSV.
  • the BRSV strains in lanes 3 and 5 (FS-l and VC-464, respectively) have a heavier F 2 protein than the BRSV strains in lanes 4 and 6 (A51908 and Md-x, respectively) .
  • BRSV strains A51908 and Md-x have an F 2 protein of the same size as that of HRSV (lane 2)
  • the F 2 protein of BRSV strains FS-l and VC-464 is smaller than that of HRSV.
  • the difference in the size of the F 2 fragment is significant because the F 2 fragment contains most of the immunogenic and neutralizing epitopes of the most important envelope protein. Therefore, like HRSV, an effective vaccine against BRSV will require incorporation of genes from viruses of both structural groups.
  • BRSV strain 391-2 which was used by Lerch et al.. supra, was isolated from an outbreak of respiratory disease in calves in North Carolina. The immunogenic and pathogenic potential, based on the characterization of the F 2 fragment, of this strain has never been reported.
  • strain A 51908 and strain FS-l of BRSV used by the present inventors, are the reference strains in the United States. Strain A 51908 has been found to cause respiratory disease and induction of neutralizing and protective antibodies in experimental infections (Mohanty et al.. J. Inf. Pis. 134. 4095 (1976)). Strain FS-l is the first isolate of BRSV in the United States, and also has been found to cause respiratory disease in calves (Smith et al.. Arch. Viral. 47. 237 (1975)).
  • BRSV Molecular cloning of BRSV genes from any strain is very difficult due to a number of attributes characteristic of BRSV replication. Some of these attributes are that: (1) BRSV has a very narrow host range; (2) BRSV yield is very low; (3) BRSV fails to depress host cell protein synthesis; and (4) BRSV is very labile.
  • the fresh virus stocks which were used were BRSV strain A 51908 (ATCC # VR-794) and BRSV strain FS-l from NVSL, U.S.D.A., Ames, Iowa. Furthermore, the maintenance medium used was Eagle's minimum essential medium with Earle's salts (MEM) .
  • MEM Eagle's minimum essential medium with Earle's salts
  • RNAs isolated from the BRSV infected cells were used to construct cDNA libraries.
  • identification of BRSV-specific clones is very difficult for the following reasons: (1) a very small proportion of the cDNA clones are virus-specific; (2) cDNA clones of HRSV do not hybridize well with mRNAs of BRSV of Northern blot hybridization; and (3) due to the pleomorphic nature of BRSV, it is difficult to purify this virus and use its genomic RNA as a probe to identify virus-specific clones.
  • BRSV-specifc clones used a number of methods to identify BRSV-specifc clones. Some of these methods are: (1) hybrid arrest and in vitro translation; (2) Northern blot hybridization; and (3) use of cDNA clones of HRSV in different hybridization conditions.
  • viral mRNA was isolated from the BRSV-infected MDBK cells by the guanidine thiocyanate-CsCl procedure (Chirgwin et al. , Biochemistry 18. 5294 (1979)). Poly(A) + -RNA was then purified using oligo (dT) cellulose (Aviv and Leder, Proc. Natl. Acad. Sci. USA 6£, 1408 (1972)), and double-stranded cDNA was synthesized by the RNase H method of Gubler and Hoffman, Gene 25, 264 (1983) .
  • the double-stranded cDNA molecules were then ligated into the EcoRI site of plasmid Bluescript (Stratagene) , and the resulting hybrid plasmids were used to transform E.coli JM109 cells using the method described by Cohen et al. , Proc. Natl. Acad. Sci. USA 69. 2110 (1972) .
  • Bacterial clones containing recombinant DNA were selected on the basis of their color and resistance to ampicillin. N-specific clones were identified by in vitro translation of mRNA obtained by hybrid-selection of randomly selected cDNA clones (Ricciardi et al.. Proc. Natl. Acad. Sci. USA 76. 4927 (1979)).
  • A60 One clone, designated A60, was selected for further analysis as it contained the largest insert among the N-specific clones. The viral specificity of this clone was further confirmed by Northern blot analysis. The cDNA clone hybridized to poly(A) + -RNA from infected cells, but not to poly(a) + -RNA from uninfected cells.
  • the nucleotide sequence of the mRNA of the N protein was obtained by sequencing the clone A60 by the dideoxynucleotide chain termination method Sanger et al.. Proc. Natl. Acad. Sci. USA 74, 5463 (1977) .
  • Clone A60 (1196 bp exclusive of poly(dA)) contained the entire N mRNA sequence lacking only 10 nucleotides of the 5' end of the mRNA.
  • the nucleotide sequence was confirmed using clone A1032 (1099 bp exclusive of poly(dA)), which expanded from nucleotide 97 to the poly(A) tail of the N mRNA.
  • nucleotide sequence of the N mRNA SEQ ID NO:2
  • deduced amino acid sequence of the N protein SEQ ID NO:3
  • the nucleotide sequence of the 5'-end untranslated region was obtained by direct sequencing of the N mRNA using a primer complementary to nucleotides 41 to 63.
  • the BRSV N mRNA contains 1196 nucleotides, excluding the poly(A) tail.
  • the mRNA has a single long open reading frame, extending from nucleotides 16 to 1188.
  • the BRSV N mRNA encodes a polypeptide of 391 amino acids with a calculated molecular weight of 42.6 kD. This value is consistent with the apparent molecular weight of 43 kD determined earlier in SDS-PAGE (Cash et al.. Virology 82. 369 (1977) and Lerch et al.. J. Virol. 63. 833 (1989)).
  • the untranslated region at the 3'-end of the BRSV N gene also shares high homology with the conserved gene end sequence present in all HRSV genes.
  • the consensus HRSV gene start sequence and gene end sequence have also been observed in other BRSV mRNAs. Thus, the presence of these consensus sequences at the start and end of each gene is believed to be a general feature of the respiratory syncytial viruses.
  • the homology at the 5'-untranslated region (exclusive of the consensus sequence) between the bovine and the human strains is nearly as high as homology between the two strains in the coding region.
  • the homology of the 5'-noncoding region is significantly higher than the homology of the coding regions.
  • the predicted amino acid sequence of the BRSV N protein was compared to that of the N protein of HRSV.
  • the N protein of BRSV is identical to that of A2 and 19537 strains of HRSV at 93% of amino acid positions. Most of the amino acid changes correspond to amino acid substitutions.
  • the apparent structure of the N protein does not seem to be affected by the amino acid changes observed. Thus, it appears that the pneumovirus N proteins are highly conserved.
  • the BRSV N gene (SEQ ID NO:2) is the preferred gene for use as a diagnostic gene probe for the detection of BRSV infection.
  • the N gene is transcribed in the largest quantity.
  • the N gene probe is the most sensitive.
  • the probe made from the N gene does not hybridize with cognate genes of other bovine respiratory viruses, but did hybridize well with RNAs extracted from different strains of BRSV.
  • Fig. 3 shows the specificity of the BRSV N gene probe for BRSV strains as opposed to other bovine respiratory viruses. There is clear hybridization of the 32 P-labeled BRSV N gene probe to the RNA of RSV strains A51908, AMES (i.e. FS-l), VC-464 and GRSV, whereas there is no hybridization with the RNA of three other bovine respiratory viruses.
  • the M gene and the P gene are also particularly useful as probes for BRSV RNA. These two genes also exhibit significant homology in various strains of BRSV. The use of nucleotide sequences as probes is fully explained in Keller and Manak, DNA Probes. Stockton Press (1989) .
  • the product of the BRSV N gene (the BRSV N protein) is also very useful as an antigen for the detection of antibodies against BRSV in serum samples.
  • BRSV antibodies are preferably detected by N gene product antigens, because the N gene product (the nucleocapsid protein) is the first protein to appear after BRSV infection (Westenbrink et al. , J. Gen. Viral 70. 591 (1989)) and is produced in the largest quantity of all the BRSV proteins produced by virus-infected cells.
  • the BRSV N protein, as well as the other BRSV proteins can be produced through recombinant means or by polymerase chain reaction (PCR) , as explained further below.
  • the BRSV proteins can also be used to produce BRSV protein antibodies.
  • a rabbit is immunized with either a naturally occurring BRSV protein or a recombinant BRSV protein.
  • a monoclonal anti-BRSV antibody is produced using conventional techniques (eth. Enzymol. , Vol. 121, Langone, J.J. and Van Vinakis, H. , Ed., Academic Press, Orlando (1986) and Roitt, I., in Essential Immunology. 5th Ed. Blackwell Scientific Publications, Boston, pp. 145-175 (1984)).
  • the anti-BRSV antibody generated is then labeled, e.g., radioactively, fluorescently or with an enzyme such as alkaline phosphatase.
  • the BRSV N gene is also useful for vaccine production because: 1) the N protein is the most abundant protein in BRSV-infected cells; 2) calves naturally infected with BRSV have high titer antibodies to N protein; and 3) N protein is necessary for cytotoxic T cell activity.
  • the production of a vaccine from the BRSV N gene or any of the other BRSV genes requires the use of the genes or fragments thereof to manufacture recombinant proteins.
  • the gene (or gene fragment) for the desired protein is operably linked to control sequences to form an expression vector. The expression vector is then used to transform a suitable host, and the transformed host, under suitable conditions, produces a recombinant form of the desired protein.
  • Recombinant forms of any of the identified BRSV proteins or fragments thereof may be produced in this manner.
  • recombinant forms of the BRSV strain A51908 nucleocapsid protein (SEQ ID NO:3), matrix protein (SEQ ID NO:5), phosphoprotein (SEQ ID NO:7), small hydrophobic protein (SEQ ID NO:9), fusion protein (SEQ ID NO:11), glycoprotein (SEQ ID NO:13) and M2 protein (SEQ ID NO:15) are produced in this manner.
  • recombinant forms of the BRSV proteins or fragments thereof of strain FS-l can also be produced in the manner set forth above.
  • the proteins or fragments thereof of strain FS-l which have been identified are the P protein (SEQ ID No:17) and the F protein (SEQ ID NO:19).
  • each of the steps for production of a recombinant protein can be done in a variety of ways.
  • the desired coding sequences can be obtained by preparing suitable cDNA from cellular messenger and manipulating the cDNA to obtain the complete sequence.
  • genomic fragments may be obtained and used directly in appropriate hosts.
  • the constructions for expression vectors operable in a variety of hosts are made using appropriate replicons and control sequences, as set forth below. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
  • control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene.
  • procaryotic, yeast, or mammalian cells are presently useful as hosts.
  • Procaryotic hosts are in general the most efficient and convenient for the production of recombinant proteins.
  • eucaryotic cells, and, in particular, mammalian cells are sometimes used for their processing capacity.
  • the preferred vectors for the BRSV genes are ba ⁇ ulovirus and IBR herpes virus.
  • suitable host cells are insect cells such as Drosophila cells, Trichoplusia ni cells (cell line TN-368) and SF-9 cells, grown maintained under conventional conditions.
  • the preferred host cells are the SF-9 cells. The culturing, maintenance and growth of insect cell lines by Agathos et al. , in Annals of the NY Acad of Sciences 589. 372 (1990) .
  • IBR herpes virus is used as the vector, the host for producing recombinant BRSV protein is a calf.
  • the procaryotes most frequently used are represented by various strains of E. coli.
  • other microbial strains may also be used, such as bacilli, for example Bacillus subtilis, various species of Pseudomonas, or other bacerial strains.
  • plasmid or bacteriophage vectors which contain replication sites and control sequences derived from a species compatible with the host are used.
  • a wide variety of vectors for many procaryotes are known (Sa brook et al. , (1989) , Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .
  • procaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems, the tryptophan (trp) promoter system and the lambda derived PL promoter and N-gene ribosome binding site, which has been made useful as a portable control cassette (U.S. Patent No. 4,711,845) .
  • any available promoter system compatible with procaryotes can be used (Sambrook et al. , supra) .
  • yeast In addition to bacteria, eucaryotic microbes, such yeast, may also be used as hosts. Laboratory strains of Saccharomyces cerevisiae. Baker's yeast, are most used, although a number of other strains are commonly available. Vectors employing the 2 micron origin of replication and, other plasmid vectors suitable for yeast expression are known (Sambrook et al. , supra) . Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes.
  • promoters known in the art include the promoter for 3-phosphoglycerate kinase, and those for other glycolytic enzymes, such as glyceraldehyde-3-phosphase dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, hosphoglycose isomerase, and glucokinase.
  • glycolytic enzymes such as glyceraldehyde-3-phosphase dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate
  • promoters which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization. (See Sambrook et al. , supra) .
  • terminator sequences are desirable at the 3' end of coding sequences. Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes. Many of the vectors illustrated contain control sequences derived from the enolase gene containing plasmid peno46 or the LEU2 gene obtained from YEpl3, however, any vector containing a yeast compatible promoter, origin or replication and other control sequences is suitable (Sambrook et al. , supra) .
  • Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and later promoters from Simian Virus 40 (SV 40) , or other viral promoters such as those derived from polyoma, adenovirus 2, bovine papilloma virus, or avian sarcoma viruses, or immunoglobulin promoters and heat shock promoters (Sambrook est gl . , supra, . General aspects of mammalian cell host system transformations have been described by Axel (U.S. Patent No. 4,399,216).
  • Enhanccer regions are important in optimizing expressin; these are, generally, sequences found upstream of the promoter region. Origins of replication may be obtained, if needed, from viral sources. However, integration into the chromosome is a common mechanism for DNA replication in eucaryotes. Plant cells are also now available as hosts, and control sequences compatible with plant cells such as the nopaline synthase promoter and polyadenylation signal sequences are available fMeth. Enzy ol.. Vol. 118, Academic Press, Orlando (1979)) .
  • transformation is done using standard techniques appropriate to such cells.
  • Such techniques include, but are not .limited to, calcium treatment employing calcium chloride for procaryotes or other cells which contain substantial cell wall barriers; infection with Agrobacterium tumefaciens for certain plant cells; calcium phosphate precipitation method for mammalian cells without cell walls; and, microprojectile bombardment for many cells including plant cells.
  • vaccines may be made by using the protein directly or by attaching a carrier to the recombinant BRSV proteins at the appropriate terminal amino acid.
  • a carrier include tetanus toxoid linked to the appropriate amino acid through tyrosine, Lipid A , hepatitis B antigen and/or a microorganism to which the protein may be linked.
  • the preferred carrier is tetanus toxoid linked to the appropriate terminal amino acid through tyrosine.
  • a conventional initial dose of such a vaccine would be 50 ⁇ g of vaccine in 0.1 ml of Freund's complete adjuvant. Conventionally, two boosts of 50 ⁇ g of vaccine in 0.1 ml of Freund's incomplete adjuvant are given at two week intervals after the initial dose.
  • the envelope proteins are also protective antigens, and the genes of these envelope proteins are also necessary for a recombinant vaccine.
  • These envelope proteins are the F protein and the G protein.
  • the M protein and the SH protein have properties which suggest that they may also be necessary for an effective vaccine.
  • the SH protein was previously thought to be a non-structural protein, the present inventors have shown that the SH protein is a third glycosylated envelope protein, in addition to the F and G proteins.
  • the nucleotide sequence of the SH gene of BRSV ID SEQ. NO:8 is significantly different from the nucleotide sequence of the SH gene of HRSV .
  • cDNA clones were constructed from intracellular poly (A + )-RNA isolated from BRSV A51908-infected cells. Recombinant DNA clones containing the M and SH genes were identified by in vitro translation of mRNA obtained by hybrid selection of randomly selected cDNA clones (Ricciardi et al. , supra) . The nucleotide sequence of the polytranscript mRNA coding for the M and SH proteins was derived from two independent clones, A564 and A22, by the dideoxynucleotide chain termination method (Sanger et al. , supra) .
  • the nucleotide sequence coding for the small hydrophobic (SH) protein is 466 nucleotides long (nucleotides 3045 to 3511) (SEQ ID NO:8).
  • the homology of the coding region, at the nucleotide level, is 45-50% depending on the HRSV strain (Table 1) .
  • the gene-start signal was identified as nucleotides 2902 to 2910 by comparison with the HRSV SH mRNA (Collins and Wertz, Virology 141. 283 (1985) and Collins et al. , J. Virol. 71 1571 (1990)).
  • the 5' untranslated region excluding the gene-start signal, is the same length as its counterpart in HRSV and shares 56% sequence identity with HRSV A2 strain but only 41% with HRSV 18537 strain.
  • the 3' untranslated region is 132 nucleotides (compared to 99 in HRSV) with a sequence homology of 50-58%.
  • the predicted SH proteins from BRSV (A51908 strain) (SEQ ID NO:9) and HRSV (A2 and 18537 strains) can be compared.
  • the predicted BRSV SH protein is 155 amino acids long. It contains a 8-amino acid extension at the carboxyl-end, relative to the HRSV SH protein.
  • BRSV SH protein has a central hydrophobic core (amino acids 14 to 41) flanked by two lysine residues (at positions 13 and 43) , which are conserved in the HRSV SH proteins (A2 and 18537 strains) .
  • This hydrophobic core contains a potential membrane-spanning region (amino acids 20 to 40) similar to the one predicted for the HRSV SH proteins (strains Al and 18537) .
  • the overall homology between the BRSV and HRSV SH proteins is surprisingly low (less than 60%) (Table 1) .
  • the amino acid identities are located mainly in the amino-end region (amino acids 1 to 23) (>65%) .
  • the central hydrophobic core (amino acids 24 to 41) has no more than 34% homology
  • the carboxyl-terminal region (amino acids 42 to 65) is highly divergent (>30% homology) (Table 1) .
  • the deduced M protein contains 316 amino acids and has a molecular weight of 28,713 daltons.
  • the BRSV M protein (SEQ ID NO:5) shares an 89% homology with the HRSV M protein , with most of the differences being due to amino acid substitutions.
  • the M protein of BRSV is moderately basic. Computer analysis predicts a single hydrophobic region (residues 188 to 204) that could act as a transmembrane domain in the BRSV M protein. The same region was predicted for the HRSV M protein (Satake and Venkateson, J. Virol. 50. 92(1984)). Comparison of HRSV M gene and BRSV M gene (SEQ ID NO:4) reveals a homology of 80% between coding regions.
  • the gene-start signal (Collins et al. , Proc- Natl. Acad. Sci. USA 82 . 4594(1988)) for the BRSV M gene (nucleotides 1 to 10) contains a single nucleotide difference at position 5 compared with the HRSV M gene-start signal, excluding the 5'-terminal nucleotide.
  • the gene-end consensus signal was identified as the sequence from nucleotide .876 to nucleotide 888.
  • the untranslated region at the 3' end is 8 nucleotides shorter in the BRSV mRNA and, as seen in other RSV genes, has a lower homology (51%) with the HRSV counterpart region (Table 1) than the coding region. As in HRSV, there is no untranslated region, other the gene-start sequence, at the 5'-end of the M mRNA of BRSV. TABLE 1
  • Intergenic region 24 % The intergenic region between M and SH genes of BRSV is 25 nucleotides long (vs 9 nucleotides in HRSV) and shares only 24% homology with the corresponding region in HRSV, suggesting that this region acts as a mere bridge between genes.
  • the polycistronic mRNA studied contains two additional open reading frames.
  • One open reading frame from nucleotides 111 to 266 encodes a protein of 52 amino acids and overlaps with the M gene.
  • a second open reading frame has also been reported for HRSV, encoding a protein of 75 amino acids, overlapping with the M gene (Satake and Venkateson, supra) .
  • HRSV high-density polycistronic mRNA was found from nucleotides 1271 to 1423 which encodes a protein of 51 amino acids. No similar protein has been described for HRSV. Since these second open reading frames are not conserved, either at the sequence level or at the relative position in the genome, they probably do not play any role in the virus replication.
  • BRSV Other strains of BRSV can be grown and their genomes cloned in accordance with the description herein.
  • the DNA sequences disclosed herein can be used as probes or to prepare degenerative primers for PCR to isolate the specific individual genes which can be cloned and utilized as described above for these additional strains of BRSV.
  • Madin-Darby bovine kidney (MDBK) cells were grow in Eagle's minimum essential medium with Earle's salts (MEM) containing 6% bovine fetal serum (BFS) .
  • MDBK Madin-Darby bovine kidney
  • BFS bovine fetal serum
  • the BRSV strain A51908 was then prepared in MDBK cell cultures propagated in MEM containing 3% BFS.
  • the viral mRNA was isolated from the BRSV-infected MDBK cells by the guanidinium thiocyanate-CsCl procedure (Chirgwin et al. , supra) , followed by two cycles of oligo (DT)-cellulose column chromotography.
  • Double-stranded cDNA was synthesized by the RNase H method of Gubler and Hoffman, supra.
  • the double-stranded cDNA molecules were ligated into the EcoRI site of plasmid Bluescript (Stratagene) .
  • the resulting hybrid plasmids were used to transform E. coli JM109 cells using the method described by Cohen et al. , supra.
  • Bacterial . clones containing recombinant DNA were selected on the basis of their color and resistance to ampicillin.
  • a cDNA clone was identified to contain sequences of the major nucleocapsid (N) gene of BRSV. This clone was used to identify other N gene clones by dot blot hydridization. One clone, designated A60, was selected for further analysis as it contained the largest insert among the N-specific clones.
  • the nucleotide sequence of the mRNA of the N protein was obtained by sequencing the clone A60 by the dideoxynucleotide chain termination method.
  • Clone A60 (1196 bp exclusive of poly(dA)) contained the entire N mRNA sequence lacking only 10 nucleotides of the 5' end of the mRNA. The nucleotide sequence was confirmed using clone A 1032 (1099 bp exclusive of poly (dA) ) , which expanded from nucleotide 97 to the poly (A) tail of the N mRNA.
  • the other BRSV genes of strain A51908 (the matrix protein gene, the phosphoprotein gene, the small hydrophobic protein gene, the fusion protein gene, the glycoprotein gene and the M2 protein gene) were isolated and sequenced using the same techniques. Furthermore, the phosphoprotein gene and the fusion protein gene of BRSV strain FS-l were also isolated and sequenced in the same manner. The sequences of the BRSV genes are set forth in the Sequence Listing as follows:
  • SEQ ID NO:2 nucleocapsid protein (N) gene
  • PCR polymerase chain reaction
  • each primer 10 copies of the BRSV genome, 200 ⁇ M of each dNTP, 2 mM MgCl 2 , 10 mM Tris-HCl (pH 8.3), and 50 mM KC1 are placed in a Perkin-Elmer Cetus Instruments Thermal Cycler. Thirty cycles of 96°C for 15 seconds, 50°C for 30 seconds and 75°C for 30 seconds are run. The BRSV N gene is then recovered using conventional techniques.
  • the other BRSV genes can also be produced using the above procedure.
  • the BRSV N gene isolated in Example 1 or produced in Example 2 is inserted into baculovirus (Autoqrapha California Nuclear Polyhedrosis Vrisu (AcNPV) (Voil et al. , J. Invertebrate Pathol. 22., 231 (1971)) using conventional techniques to form an expression vector.
  • the expression vector is then used to infect Spodoptera frugiperda (SF-9) cells.
  • the SF-9 cells are maintained in a serum-free/protein-free medium developed by Maiorella et al. , Bio/Technology .6, 1406 (1988).
  • the serum-free/ protein-free medium is composed of IPL/41 medium (JR Scientific, Woodland, CA) supplemented with tryptose phosphate broth (Oxoid USA, Columbia, MD) , fetal bovine serum (Gibco, Grand Island, NY) , and pluronic polyol F68 (BASF Wyandotte, Porsippamy, NJ) .
  • the SF-9 cells After infection with the baculovirus expression vector, the SF-9 cells are grown in a medium composed of IPL/41 medium supplemented with cod liver oil polyunsaturated fatty acid methyl esters, cholesterol, alpha-tocopherol acetate, Tween 80 and diluted Yestolate (Difco) .
  • the cells are grown in spinner flasks, stirred at 75-100 rpm, at 27°C with an air atmosphere.
  • BRSV N protein titre peaks as cell lysis begins. SDS-PAGE analysis is then used to identify and isolate the recombinant BRSV N protein.
  • the other BRSV genes from Example 1 may be used in the same procedure described above to isolate and identify their respective recombinant proteins.
  • ADDRESSEE Venable, Baetjer, Howard & Civiletti
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI- SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • CAACTGTTAT CAACCAGCAA ATATACTATT CAACGTAGTA CAGGTGACAA CATTGATATA 120
  • CTGCAAATCA AGTTCATATC AGAAAGCCTT TGGTAAGCTT CAAAGAAGAA CTGCCATCAA 1500
  • GCATCCAATC AAGCACAGCA CACACCGGAC ACTCCTTGAA TCCACCAGCT GGTTGAACTT 3120
  • GCAACCCCCC CGAAAACCAC CAAGACCACA ACAACTCCCA AACACTCCCT CATGTGCCCT 4080
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • GGC AAT ATA GAA ATA GAG TCA AGG AAG TCT TAC AAA AAG ATG CTA AAA 435 Gly Asn He Glu He Glu Ser Arg Lys Ser Tyr Lys Lys Met Leu Lys 125 130 135 140
  • GGT ATG ATA GTG CTA TGT GTT GCT GCT TTG GTT ATA ACA AAA TTA GCA 531 Gly Met He Val Leu Cys Val Ala Ala Leu Val He Thr Lys Leu Ala 160 165 170
  • GTA CTA AGG AAT GAA ATG AAA CAA TAC AAA GGA CTT ATC CCG AAA GAT 627 Val Leu Arg Asn Glu Met Lys Gin Tyr Lys Gly Leu He Pro Lys Asp 190 195 200
  • GCT GCC AAA GCA TAT GCG GAA CAA TTA AAA GAG AAT GGG GTC ATC AAT 1107 Ala Ala Lys Ala Tyr Ala Glu Gin Leu Lys Glu Asn Gly Val He Asn 350 355 360
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • GTA ACT GAC AAT AAA GGG GCA TTC AAG TAC ATT AAA CCA CAA AGT CAA 672 Val Thr Asp Asn Lys Gly Ala Phe Lys Tyr He Lys Pro Gin Ser Gin 210 215 220
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • MOLECULE TYPE protein
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine resoiratory syncytial virus
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • GTA CTA CAC TTG GAG GGA GAG GTG AAC AAA ATT AAA AAT GCA CTG CTA 529 Val Leu His Leu Glu Gly Glu Val Asn Lys He Lys Asn Ala Leu Leu 160 165 170
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • CAC AGA ACC AGC CCT GAA GCC AAA CTG CAA ACC AAA AAA AAC ACG GCA 723 His Arg Thr Ser Pro Glu Ala Lys Leu Gin Thr Lys Lys Asn Thr Ala 225 230 235
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • AAA AAG ACC ATC AAG AAC ACA ATA GAT ATT CAC AAC GAA ATA AAT GGT 528 Lys Lys Thr He Lys Asn Thr He Asp He His Asn Glu He Asn Gly 160 165 170
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • ORGANISM Bovine respiratory syncytial virus
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Bovine respiratory syncytial virus
  • GAGCTTCTAC CTAAAGTTAA CAATCATGAT TGTAGGATAT CCAACATAGG AACTGTGATA 660
  • GAATTCCAAC AAAAAAACAA TAGATTGTTA GAAATTGCTA GGGAATTTAG TGTAAATGCT 720
  • GGTATTACCA CACCCCTCAG TACATACATG TTGACCAATA GTGAATTACT ATCACTAATT 780
  • ORGANISM Bovine respiratory syncytial virus
  • STRAIN FS-l

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Abstract

The present invention relates to genes derived from bovine respiratory syncytial virus (BRSV). The invention also relates to vectors produced with the genes, expression systems for the genes, as well as diagnostic probes comprising the genes, recombinant proteins and vaccines. The recombinant nucleocapsid protein is particularly useful for diagnostic testing for early detection of a BRSV infection.

Description

BOVINE RESPIRATORY SYNCYTIAL VIRUS GENES
RELATED APPLICATIONS
This is a continuation-in-part of United States patent application Serial No. 07/608,937, filed November 5, 1990.
BACKGROUND OF THE INVENTION
Bovine Respiratory Syncytial Virus (BRSV) is an RNA virus which is recognized as an important cause of lower respiratory disease of cattle in Asia, Europe and the United States. In cattle, BRSV infection is more significantly associated with respiratory disease than any other virus. Furthermore, the highest incidence of severe BRSV disease in cattle is between 2 and 4.5 months of age. About 70 percent of cattle have been infected by BRSV by 9 months of age.
The characteristics of BRSV infection include extensive damage to the mucuous membranes, leaving the respiratory tract susceptible to dust, debris and secondary infectious agents. BRSV infection also causes a condition which resembles systemic anaphylaxis or hypersensitivity. Outbreaks of BRSV infection usually last about 10 to 14 days, and are characterized by high mortality.
However, BRSV is not easily grown, is closely associated with the cell membrane and is inherently unstable (Stott and Taylor, Arch. Viroloσv 84. 1 (1985)). Therefore, purification of the virus by biophysical techniques is extremely difficult.
Antibodies to BRSV were first found in cattle sera in 1968, and in 1970, the virus was isolated. BRSV, human respiratory syncytial virus (HRSV) and pneumonia virus of mice comprise the genus Pneumovirus which is within the family Paramyxoviridae.
Viruses of this family have enveloped pleomorphic virions which contain helical, elongated nucleocapsids. The genome is linear, single-stranded RNA which replicates in the cytoplasm.
Morphologically, the BRSV virion appears as round or pleomorphic forms which measure about 80 to 500 mm across, or as filamentous forms up to several um in length. The outer membrane of the virion is studded with projections about 12 mm long, each of which is about 10 mm apart.
Purified virions contain a unique species of single-stranded RNA of which at least 93 percent is negative sense. Discrepancies regarding the size and number of polypeptides in the virion are due, in part, to differences between virus strains (Stott and Taylor, supra) .
Two small polypeptides have been detected. The smaller polypeptide has a molecular weight between about 10,000 and 13,000 daltons, and the. larger polypeptide has a molecular weight between about 19,000 and 25,000 daltons. The large polypeptide is a non-glycosylated protein. Neither of the small polypeptides has any known functions (Stott and Taylor, supra) .
A larger protein, the M protein (about 27,000 to 28,000 daltons) is believed to be the membrane protein. The M protein has 256 amino acids, and is relatively basic with two hydrophobic regions in the C-terminal third of the protein.
The P protein is a phosphorylated protein with a molecular weight of about 32,000 to 38,000 daltons. This protein is associated with the nucleocapsid.
However, the nucleocapsids isolated from purified BRSV contain primarily NP protein in association with RNA. The NP protein has a molecular weight of between about 40,000 and 44,000 daltons, and has 467 amino acids, most of which are basic amino acids.
Two glycoproteins which are believed to be located on the surface of the virion are the F protein and G protein. The F protein has a molecular weight of between about 66,000 and 68,000 daltons, and the G protein has a molecular weight of between about 79,000 and 90,000 daltons. These two proteins have a rod-shaped morphology, suggesting that they may be the studded projections of the virion.
The F protein is comprised of two smaller glycoproteins linked by disulfide bonds. One of the smaller glycoprotein has a molecular weight between about 43,000 and 56,000 daltons and the other smaller glycoprotein has a molecular weight between about 19,000 and 22,000 daltons. The F protein has been shown to be the fusion protein by the inhibition of cell fusion by a monoclonal antibody to the F protein.
The G protein is believed to be the attachment protein of the virion. Monoclonal antibodies to either the F protein or the G protein neutralize infectivity of the virus.
There is little known about the largest polypeptide, the L protein. This protein has a molecular weight of between about 160,000 daltons and 200,000 daltons and is believed to be the RNA polymerase of the virion.
Although BRSV and HRSV differ in plaque reduction tests using bovine sera, cattle have been reported to be equally protected from BRSV infection by either BRSV or HRSV antibodies. Any differences detected by neutralization are believed to reflect changes in the epitopes on either the F protein or G protein (Stott and Taylor, supra) . Furthermore, two monoclonal antibodies to the BRSV fusion protein appear to react complement fixation and immunofluoresence tests, all strains of respiratory syncytial virus cross-react (Stott and Taylor, supra) .
Regarding respiratory syncytial virus vaccines, three different vaccines for HRSV have been tested, but found not to provide acceptable immunization against HRSV infection (Stott and Taylor, supra) . Specifically, the three vaccines which have been tested are: 1) an inactivated antigen combined with adjuvant given intramuscularly; 2) live attenuated viruses given intranasally; and 3) live modified virus given intramuscularl .
Furthermore, the intramuscular vaccination of cattle with a live mutant strain of HRSV does not confer immunity to the cattle (Stott and Taylor, supra) . However, cattle have been protected against BRSV infection by intramuscular vaccination with glutaraldehyde-fixed, respiratory syncytial virus infected bovine nasal mucosa cells (Stott and Taylor, supra) .
Another experimental attenuated BRSV vaccine has been reported to be successful by Bohlender et al. In the August, 1982 issue of Modern Veterinary Practice at page 613. This vaccine was found to reduce the average morbidity among calves receiving the vaccine by about half.
Although BRSV and HRSV have traditionally been classified in the same genus, Pneumovirus. as stated above, at least one group of researchers have suggested that these two viruses be classified in separate groups of the genus (Lerch et al., J. Virol. 64, 5559 (1990)). A comparison of the amino acid sequences of the G protein of BRSV with the G protein of either subgroup A or B of HRSV showed only a 29 to 30% amino acid identity. Furthermore, antisera to the BRSV G protein, made by using a recombinant vector to immunize animals, recognized the BRSV G protein but not the HRSV G protein, and vice versa (Lerch et al. , supra) .
As is evident from the seriousness of BRSV infection, a rapid diagnostic test for this virus, and a vaccine which would prevent such infection are greatly needed. However, there are no rapid diagnostic tests, and the known vaccines have been ineffective and/or difficult to produce. Particularly, live attenuated BRSV vaccines are unstable, and killed vaccines have been found to be unsatisfactory because the inactivation process probably alters immunogenic epitopes on the virion surface.
SUMMARY OF THE INVENTION
Recognizing the severity of BRSV infection, the present invention relates to genes derived from BRSV, vectors produced with the genes and expression systems for the genes. The genes comprise the nucleotide sequences set forth as in the Sequence Listing as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6; SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14; SEQ ID NO:16, and SEQ ID NO:18. The invention further relates to fragments of these genes.
The invention also encompasses diagnostic probes comprising these genes or fragments thereof. All of the genes or their fragments are useful as diagnostic probes.
The invention further encompasses purified BRSV proteins or fragments thereof. These purified proteins and fragments can be used to detect BRSV antibodies, and can be used as vaccines. The purified proteins which are useful for the detection of BRSV antibodies and as vaccines are proteins set forth in the Sequence Listing as SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:ll, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:19. However, the protein of SEQ ID NO:3 is particularly useful for diagnostic testing for the presence of BRSV antibodies.
A further aspect of the invention are antibodies to the gene products or fragments thereof.
BRIEF DESCRIPTION OF THE FIGURES
The various objects, advantages and novel features of the invention will be more readily appreciated from the following detailed description when read in conjunction with the appended figures in which:
Fig. 1 shows the genetic map of BRSV strain A2;
Fig. 2 shows an SDS-PAGE gel in which [3H] glucosamine labeled antigens were immunoprecipitated with BRSV (strain A51908) antiserum; and
Fig. 3 shows an autoradiograph of the hybridization of 3 P-labeled BRSV-N gene probe to the RNA of a variety of bovine respiratory viruses.
DETAILED DESCRIPTION OF THE INVENTION
In order to provide a clear and consistent understanding of the specification and the claims, the following definitions are provided:
Adjacent: A position in a nucleotide sequence immediately 5' or 3' to a defined sequence.
Cell Culture: A proliferating mass of cells which may be in an undifferentiated or differentiated state. As used herein "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. Thus "transformants" or "transformed cells" includes the primary subject cell and cultures derived therefrom without regard for the number of tranfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny which have the same functionality as sreened for in the originally transformed cell, are included. Where distinct designations are intended, it will be clear from the context.
Coding Sequence: A deoxyribonucleotide sequence which when transcribed and translated results in the formation of a cellular protein, or a ribonucleotide sequence which when translated results in the formation of a cellular protein.
Control Sequences: Refers to DNA sequences necessary for the expression of an operablylinked coding sequence in a particular host organism. The control sequences which are suitable for procaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood, sequences. Eucaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
Expression System: Refers to DNA sequences containing a desired coding sequence and control sequences in operable linkage, so that hosts transformed with these sequences are capable of producing the encoded proteins. In order to effect transformation, the expression system may be included on a vector; however, the relevant DNA may then also be integrated into the host chromosome.
Gene: A discrete nucleic acid region which is responsible for a discrete cellular product.
Operably Linked: Refers to juxtaposition such that the normal function of the components can be performed. Thus, a coding sequence "operably linked" to control equences refers to a configuratin wherein the coding sequence cn be expressed under the control of these sequences.
Promoter: The 5'- flanking, non-coding sequence adjacent a coding sequence which is involved in the initiation of transcription of the coding sequence.
Substantial Sequence Homology: Donates nucleotide sequences that are substantially functionally equivalent to one another. Nucleotide differences between such sequences having substantial sequence homology will be de minimus in affecting the function of the gene products or an RNA coded for such a sequence.
With the above definitions in mind, the present invention relates to the novel genes and gene fragments derived from BRSV. The entire genome for BRSV strain A 51908 is presented in SEQ ID NO:l. The genes and gene fragments thereof are:
1. the nucleocapsid protein gene (the N gene) (SEQ ID NO:2) ;
2. the matrix protein gene (the M gene) (SEQ ID NO:4) ; 3. the phosphoprotein gene (the P gene) (SEQ ID NO:6) ;
4. the small hydrophobic protein gene (the SH gene) (SEQ ID NO:8) ;
5. the fusion protein gene (the F gene) (SEQ ID NO:10) ;
6. the glycoprotein gene (the G gene) (SEQ ID NO:12) ; and
7. the M2 protein gene (SEQ ID NO:14).
The position of these genes in the BRSV genome is shown in the gene map of Fig. 1.
These genes were derived from two strains of BRSV. The genes designated by SEQ ID NOS. 2, 4, 6, 8, 10, 12 and 14 above were derived from BRSV strain A 51908, which is available from the American Type * Culture Collection (ATCC #VR-794) . The other strain is strain FS-l, which is available from the National Veterinary Services Laboratory (NVSL) U.S.D.A., Ames, Iowa. Only the P gene and F gene have been isolated and sequenced from BRSV strain FS-l. The P gene of BRSV strain FS-l is set forth in the Sequence Listing as SEQ ID No:16, and the F gene of this strain is set forth as SEQ ID NO:18.
As has been recognized in HRSV, there are distinct differences between strains, and knowledge of these differences is necessary for the understanding of clinical and epidemiological characteristics, as well as for vaccine development. With the use of polyclonal and monoclonal antibodies, two distinct antigenic subgroups (A&B) of HRSV have been recognized. It has been found that viruses of both subgroups circulate in nature, and that for a vaccine to be effective, viruses from both the subgroups must be included. Both antigenic and structural differences between subgroups have been described for several HRSV proteins. Distinct differences in the size of the homologous fusion (F) protein cleavage products and phosphoprotein (P) of subgroup A and B viruses have been demonstrated.
In contrast, to HRSV, antigenic and structural comparisons among strains of BRSV have not, until now, been reported. Recently, Lerch et al.. compared BRSV strain 391-2 with HRSV strain A (Lerch et al. , J. Virol. 63, 833 (1989)). The most striking difference was found in the F2 fragment. The F2 fragment of BRSV strain 391-2 had a lower molecular weight than the F fragment of HRSV.
However, the present inventors have compared the polypeptides of four strains of BRSV. Based on the size of the F2 fragment and P protein the BRSV strains examined could be classified into groups.
Fig. 2 shows the different migration patterns of the F2 protein of different strains of BRSV. Particularly, lanes 3-6 show four different strains of BRSV. The BRSV strains in lanes 3 and 5 (FS-l and VC-464, respectively) have a heavier F2 protein than the BRSV strains in lanes 4 and 6 (A51908 and Md-x, respectively) . Furthermore, BRSV strains A51908 and Md-x have an F2 protein of the same size as that of HRSV (lane 2) , whereas the F2 protein of BRSV strains FS-l and VC-464 is smaller than that of HRSV.
The difference in the size of the F2 fragment is significant because the F2 fragment contains most of the immunogenic and neutralizing epitopes of the most important envelope protein. Therefore, like HRSV, an effective vaccine against BRSV will require incorporation of genes from viruses of both structural groups.
BRSV strain 391-2 which was used by Lerch et al.. supra, was isolated from an outbreak of respiratory disease in calves in North Carolina. The immunogenic and pathogenic potential, based on the characterization of the F2 fragment, of this strain has never been reported.
In contrast, strain A 51908 and strain FS-l of BRSV, used by the present inventors, are the reference strains in the United States. Strain A 51908 has been found to cause respiratory disease and induction of neutralizing and protective antibodies in experimental infections (Mohanty et al.. J. Inf. Pis. 134. 4095 (1976)). Strain FS-l is the first isolate of BRSV in the United States, and also has been found to cause respiratory disease in calves (Smith et al.. Arch. Viral. 47. 237 (1975)).
Molecular cloning of BRSV genes from any strain is very difficult due to a number of attributes characteristic of BRSV replication. Some of these attributes are that: (1) BRSV has a very narrow host range; (2) BRSV yield is very low; (3) BRSV fails to depress host cell protein synthesis; and (4) BRSV is very labile.
Therefore, a number of cell lines and growth conditions were examined before the optimal conditions for maximum virus yield were determined. Madin Darby bovine kidney (MDBK) cells, 3% bovine fetal serum in maintenance medium, fresh virus stocks without freezing and thawing, and harvest at 48 hours after infection were found to be the optimal growth conditions. It was also found that treatment with Actinomycin D at a concentration of 2.5 μg/ml for 4 hours prior to harvesting cells for mRNAs inhibited some host cell mRNA synthesis. However, higher concentrations or long periods of treatment with Actinomycin D were toxic to MDBK cells.
As noted above, the fresh virus stocks which were used were BRSV strain A 51908 (ATCC # VR-794) and BRSV strain FS-l from NVSL, U.S.D.A., Ames, Iowa. Furthermore, the maintenance medium used was Eagle's minimum essential medium with Earle's salts (MEM) .
The mRNAs isolated from the BRSV infected cells were used to construct cDNA libraries. However, identification of BRSV-specific clones is very difficult for the following reasons: (1) a very small proportion of the cDNA clones are virus-specific; (2) cDNA clones of HRSV do not hybridize well with mRNAs of BRSV of Northern blot hybridization; and (3) due to the pleomorphic nature of BRSV, it is difficult to purify this virus and use its genomic RNA as a probe to identify virus-specific clones.
However, the present inventors used a number of methods to identify BRSV-specifc clones. Some of these methods are: (1) hybrid arrest and in vitro translation; (2) Northern blot hybridization; and (3) use of cDNA clones of HRSV in different hybridization conditions.
In identifying the BRSV specific clones, viral mRNA was isolated from the BRSV-infected MDBK cells by the guanidine thiocyanate-CsCl procedure (Chirgwin et al. , Biochemistry 18. 5294 (1979)). Poly(A)+-RNA was then purified using oligo (dT) cellulose (Aviv and Leder, Proc. Natl. Acad. Sci. USA 6£, 1408 (1972)), and double-stranded cDNA was synthesized by the RNase H method of Gubler and Hoffman, Gene 25, 264 (1983) .
The double-stranded cDNA molecules were then ligated into the EcoRI site of plasmid Bluescript (Stratagene) , and the resulting hybrid plasmids were used to transform E.coli JM109 cells using the method described by Cohen et al. , Proc. Natl. Acad. Sci. USA 69. 2110 (1972) . Bacterial clones containing recombinant DNA were selected on the basis of their color and resistance to ampicillin. N-specific clones were identified by in vitro translation of mRNA obtained by hybrid-selection of randomly selected cDNA clones (Ricciardi et al.. Proc. Natl. Acad. Sci. USA 76. 4927 (1979)).
One clone, designated A60, was selected for further analysis as it contained the largest insert among the N-specific clones. The viral specificity of this clone was further confirmed by Northern blot analysis. The cDNA clone hybridized to poly(A)+-RNA from infected cells, but not to poly(a)+-RNA from uninfected cells.
The nucleotide sequence of the mRNA of the N protein was obtained by sequencing the clone A60 by the dideoxynucleotide chain termination method Sanger et al.. Proc. Natl. Acad. Sci. USA 74, 5463 (1977) . Clone A60 (1196 bp exclusive of poly(dA)) contained the entire N mRNA sequence lacking only 10 nucleotides of the 5' end of the mRNA. The nucleotide sequence was confirmed using clone A1032 (1099 bp exclusive of poly(dA)), which expanded from nucleotide 97 to the poly(A) tail of the N mRNA.
The nucleotide sequence of the N mRNA (SEQ ID NO:2) and deduced amino acid sequence of the N protein (SEQ ID NO:3) of BRSV are shown in the Sequence Listing. The nucleotide sequence of the 5'-end untranslated region was obtained by direct sequencing of the N mRNA using a primer complementary to nucleotides 41 to 63.
The BRSV N mRNA contains 1196 nucleotides, excluding the poly(A) tail. The mRNA has a single long open reading frame, extending from nucleotides 16 to 1188. The BRSV N mRNA encodes a polypeptide of 391 amino acids with a calculated molecular weight of 42.6 kD. This value is consistent with the apparent molecular weight of 43 kD determined earlier in SDS-PAGE (Cash et al.. Virology 82. 369 (1977) and Lerch et al.. J. Virol. 63. 833 (1989)).
Comparison of the nucleotide sequence of the BRSV N gene (SEQ ID NO:2) with the published sequence of HRSV N gene revealed an overall homology of 81%. It is interesting to note the high homology with the conserved gene start sequence present in all mRNAs of HRSV.
Additionally, the untranslated region at the 3'-end of the BRSV N gene (SEQ ID NO:2) also shares high homology with the conserved gene end sequence present in all HRSV genes. The consensus HRSV gene start sequence and gene end sequence have also been observed in other BRSV mRNAs. Thus, the presence of these consensus sequences at the start and end of each gene is believed to be a general feature of the respiratory syncytial viruses.
It was also noted that the homology at the 5'-untranslated region (exclusive of the consensus sequence) between the bovine and the human strains is nearly as high as homology between the two strains in the coding region. However, between human strains, the homology of the 5'-noncoding region is significantly higher than the homology of the coding regions.
The predicted amino acid sequence of the BRSV N protein (SEQ ID NO:3) was compared to that of the N protein of HRSV. The N protein of BRSV is identical to that of A2 and 19537 strains of HRSV at 93% of amino acid positions. Most of the amino acid changes correspond to amino acid substitutions. The apparent structure of the N protein does not seem to be affected by the amino acid changes observed. Thus, it appears that the pneumovirus N proteins are highly conserved.
The BRSV N gene (SEQ ID NO:2) is the preferred gene for use as a diagnostic gene probe for the detection of BRSV infection. Of all the genes transcribed in BRSV infected cells, the N gene is transcribed in the largest quantity. Also, of the three internal protein genes (N, P and M) tested as probes, the N gene probe is the most sensitive. Furthermore, the probe made from the N gene does not hybridize with cognate genes of other bovine respiratory viruses, but did hybridize well with RNAs extracted from different strains of BRSV.
Fig. 3 shows the specificity of the BRSV N gene probe for BRSV strains as opposed to other bovine respiratory viruses. There is clear hybridization of the 32P-labeled BRSV N gene probe to the RNA of RSV strains A51908, AMES (i.e. FS-l), VC-464 and GRSV, whereas there is no hybridization with the RNA of three other bovine respiratory viruses.
The M gene and the P gene are also particularly useful as probes for BRSV RNA. These two genes also exhibit significant homology in various strains of BRSV. The use of nucleotide sequences as probes is fully explained in Keller and Manak, DNA Probes. Stockton Press (1989) .
The product of the BRSV N gene (the BRSV N protein) is also very useful as an antigen for the detection of antibodies against BRSV in serum samples. BRSV antibodies are preferably detected by N gene product antigens, because the N gene product (the nucleocapsid protein) is the first protein to appear after BRSV infection (Westenbrink et al. , J. Gen. Viral 70. 591 (1989)) and is produced in the largest quantity of all the BRSV proteins produced by virus-infected cells. The BRSV N protein, as well as the other BRSV proteins (the M, P, SH, F, G, and M2 proteins) can be produced through recombinant means or by polymerase chain reaction (PCR) , as explained further below.
The BRSV proteins can also be used to produce BRSV protein antibodies. In order to produce an anti-BRSV antibody, a rabbit is immunized with either a naturally occurring BRSV protein or a recombinant BRSV protein. Alternatively, a monoclonal anti-BRSV antibody is produced using conventional techniques ( eth. Enzymol. , Vol. 121, Langone, J.J. and Van Vinakis, H. , Ed., Academic Press, Orlando (1986) and Roitt, I., in Essential Immunology. 5th Ed. Blackwell Scientific Publications, Boston, pp. 145-175 (1984)). The anti-BRSV antibody generated is then labeled, e.g., radioactively, fluorescently or with an enzyme such as alkaline phosphatase.
In addition to being the preferred gene for diagnostic purposes, the BRSV N gene is also useful for vaccine production because: 1) the N protein is the most abundant protein in BRSV-infected cells; 2) calves naturally infected with BRSV have high titer antibodies to N protein; and 3) N protein is necessary for cytotoxic T cell activity. The production of a vaccine from the BRSV N gene or any of the other BRSV genes requires the use of the genes or fragments thereof to manufacture recombinant proteins. In order to produce a recombinant protein, the gene (or gene fragment) for the desired protein is operably linked to control sequences to form an expression vector. The expression vector is then used to transform a suitable host, and the transformed host, under suitable conditions, produces a recombinant form of the desired protein.
Recombinant forms of any of the identified BRSV proteins or fragments thereof may be produced in this manner. Particularly, recombinant forms of the BRSV strain A51908 nucleocapsid protein (SEQ ID NO:3), matrix protein (SEQ ID NO:5), phosphoprotein (SEQ ID NO:7), small hydrophobic protein (SEQ ID NO:9), fusion protein (SEQ ID NO:11), glycoprotein (SEQ ID NO:13) and M2 protein (SEQ ID NO:15) are produced in this manner.
Furthermore, recombinant forms of the BRSV proteins or fragments thereof of strain FS-l can also be produced in the manner set forth above. The proteins or fragments thereof of strain FS-l which have been identified are the P protein (SEQ ID No:17) and the F protein (SEQ ID NO:19).
Each of the steps for production of a recombinant protein can be done in a variety of ways. For example, the desired coding sequences can be obtained by preparing suitable cDNA from cellular messenger and manipulating the cDNA to obtain the complete sequence. Alternatively, genomic fragments may be obtained and used directly in appropriate hosts. The constructions for expression vectors operable in a variety of hosts are made using appropriate replicons and control sequences, as set forth below. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors.
The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene. Generally, procaryotic, yeast, or mammalian cells are presently useful as hosts. Procaryotic hosts are in general the most efficient and convenient for the production of recombinant proteins. However, eucaryotic cells, and, in particular, mammalian cells are sometimes used for their processing capacity.
The preferred vectors for the BRSV genes are baσulovirus and IBR herpes virus. When baculovirus is used as the vector, suitable host cells are insect cells such as Drosophila cells, Trichoplusia ni cells (cell line TN-368) and SF-9 cells, grown maintained under conventional conditions. The preferred host cells are the SF-9 cells. The culturing, maintenance and growth of insect cell lines by Agathos et al. , in Annals of the NY Acad of Sciences 589. 372 (1990) . When IBR herpes virus is used as the vector, the host for producing recombinant BRSV protein is a calf.
In addition to the preferred insect host cells, the procaryotes most frequently used are represented by various strains of E. coli. However, other microbial strains may also be used, such as bacilli, for example Bacillus subtilis, various species of Pseudomonas, or other bacerial strains. In such procaryotic systems, plasmid or bacteriophage vectors which contain replication sites and control sequences derived from a species compatible with the host are used. A wide variety of vectors for many procaryotes are known (Sa brook et al. , (1989) , Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) . Commonly used procaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems, the tryptophan (trp) promoter system and the lambda derived PL promoter and N-gene ribosome binding site, which has been made useful as a portable control cassette (U.S. Patent No. 4,711,845) . However, any available promoter system compatible with procaryotes can be used (Sambrook et al. , supra) .
In addition to bacteria, eucaryotic microbes, such yeast, may also be used as hosts. Laboratory strains of Saccharomyces cerevisiae. Baker's yeast, are most used, although a number of other strains are commonly available. Vectors employing the 2 micron origin of replication and, other plasmid vectors suitable for yeast expression are known (Sambrook et al. , supra) . Control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes. Additional promoters known in the art include the promoter for 3-phosphoglycerate kinase, and those for other glycolytic enzymes, such as glyceraldehyde-3-phosphase dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, hosphoglycose isomerase, and glucokinase. Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and enzymes responsible for maltose and galactose utilization. (See Sambrook et al. , supra) .
It is also believed that terminator sequences are desirable at the 3' end of coding sequences. Such terminators are found in the 3' untranslated region following the coding sequences in yeast-derived genes. Many of the vectors illustrated contain control sequences derived from the enolase gene containing plasmid peno46 or the LEU2 gene obtained from YEpl3, however, any vector containing a yeast compatible promoter, origin or replication and other control sequences is suitable (Sambrook et al. , supra) .
It is also, possible to express genes encoding polypeptides in eucaryotic host cell cultures derived from multicellular organisms (Meth. Enzvmol.. Vol 58, Academic Press, Orlando (1979)). Useful host cell lines include murine myelomas N51, Vero and HeLa cells, and Chinese hamster ovary (CHO) cells. Expression vectors for such cells ordinarily include promoters and control sequences compatible with mammalian cells such as, for example, the commonly used early and later promoters from Simian Virus 40 (SV 40) , or other viral promoters such as those derived from polyoma, adenovirus 2, bovine papilloma virus, or avian sarcoma viruses, or immunoglobulin promoters and heat shock promoters (Sambrook est gl . , supra, . General aspects of mammalian cell host system transformations have been described by Axel (U.S. Patent No. 4,399,216).
It is now also appears that "enhancer" regions are important in optimizing expressin; these are, generally, sequences found upstream of the promoter region. Origins of replication may be obtained, if needed, from viral sources. However, integration into the chromosome is a common mechanism for DNA replication in eucaryotes. Plant cells are also now available as hosts, and control sequences compatible with plant cells such as the nopaline synthase promoter and polyadenylation signal sequences are available fMeth. Enzy ol.. Vol. 118, Academic Press, Orlando (1979)) .
Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. Such techniques include, but are not .limited to, calcium treatment employing calcium chloride for procaryotes or other cells which contain substantial cell wall barriers; infection with Agrobacterium tumefaciens for certain plant cells; calcium phosphate precipitation method for mammalian cells without cell walls; and, microprojectile bombardment for many cells including plant cells.
Following production of the recombinant proteins, vaccines may be made by using the protein directly or by attaching a carrier to the recombinant BRSV proteins at the appropriate terminal amino acid. Useful carriers include tetanus toxoid linked to the appropriate amino acid through tyrosine, Lipid A , hepatitis B antigen and/or a microorganism to which the protein may be linked. The preferred carrier is tetanus toxoid linked to the appropriate terminal amino acid through tyrosine.
A conventional initial dose of such a vaccine would be 50 μg of vaccine in 0.1 ml of Freund's complete adjuvant. Conventionally, two boosts of 50 μg of vaccine in 0.1 ml of Freund's incomplete adjuvant are given at two week intervals after the initial dose.
In addition to the N protein, the envelope proteins are also protective antigens, and the genes of these envelope proteins are also necessary for a recombinant vaccine. These envelope proteins are the F protein and the G protein.
Furthermore, the M protein and the SH protein have properties which suggest that they may also be necessary for an effective vaccine. Although the SH protein was previously thought to be a non-structural protein, the present inventors have shown that the SH protein is a third glycosylated envelope protein, in addition to the F and G proteins. Furthermore, the nucleotide sequence of the SH gene of BRSV (ID SEQ. NO:8) is significantly different from the nucleotide sequence of the SH gene of HRSV .
In order to identify the M gene and the SH gene, cDNA clones were constructed from intracellular poly (A+)-RNA isolated from BRSV A51908-infected cells. Recombinant DNA clones containing the M and SH genes were identified by in vitro translation of mRNA obtained by hybrid selection of randomly selected cDNA clones (Ricciardi et al. , supra) . The nucleotide sequence of the polytranscript mRNA coding for the M and SH proteins was derived from two independent clones, A564 and A22, by the dideoxynucleotide chain termination method (Sanger et al. , supra) .
The nucleotide sequence coding for the small hydrophobic (SH) protein is 466 nucleotides long (nucleotides 3045 to 3511) (SEQ ID NO:8). The homology of the coding region, at the nucleotide level, is 45-50% depending on the HRSV strain (Table 1) . The gene-start signal was identified as nucleotides 2902 to 2910 by comparison with the HRSV SH mRNA (Collins and Wertz, Virology 141. 283 (1985) and Collins et al. , J. Virol. 71 1571 (1990)). The 5' untranslated region, excluding the gene-start signal, is the same length as its counterpart in HRSV and shares 56% sequence identity with HRSV A2 strain but only 41% with HRSV 18537 strain. The 3' untranslated region is 132 nucleotides (compared to 99 in HRSV) with a sequence homology of 50-58%.
The predicted SH proteins from BRSV (A51908 strain) (SEQ ID NO:9) and HRSV (A2 and 18537 strains) can be compared. The predicted BRSV SH protein is 155 amino acids long. It contains a 8-amino acid extension at the carboxyl-end, relative to the HRSV SH protein.
Computer analysis predicts that BRSV SH protein has a central hydrophobic core (amino acids 14 to 41) flanked by two lysine residues (at positions 13 and 43) , which are conserved in the HRSV SH proteins (A2 and 18537 strains) . This hydrophobic core contains a potential membrane-spanning region (amino acids 20 to 40) similar to the one predicted for the HRSV SH proteins (strains Al and 18537) . There are three potential N-glycosylation sites at positions 2, 3 and 52, but only the one at position 3 is conserved in all three proteins.
The overall homology between the BRSV and HRSV SH proteins is surprisingly low (less than 60%) (Table 1) . The amino acid identities are located mainly in the amino-end region (amino acids 1 to 23) (>65%) . However, the central hydrophobic core (amino acids 24 to 41) has no more than 34% homology, and the carboxyl-terminal region (amino acids 42 to 65) is highly divergent (>30% homology) (Table 1) . The deduced M protein contains 316 amino acids and has a molecular weight of 28,713 daltons. The BRSV M protein (SEQ ID NO:5) shares an 89% homology with the HRSV M protein , with most of the differences being due to amino acid substitutions.
In relation to the M protein of HRSV, the M protein of BRSV is moderately basic. Computer analysis predicts a single hydrophobic region (residues 188 to 204) that could act as a transmembrane domain in the BRSV M protein. The same region was predicted for the HRSV M protein (Satake and Venkateson, J. Virol. 50. 92(1984)). Comparison of HRSV M gene and BRSV M gene (SEQ ID NO:4) reveals a homology of 80% between coding regions.
The gene-start signal (Collins et al. , Proc- Natl. Acad. Sci. USA 82. 4594(1988)) for the BRSV M gene (nucleotides 1 to 10) contains a single nucleotide difference at position 5 compared with the HRSV M gene-start signal, excluding the 5'-terminal nucleotide. The gene-end consensus signal was identified as the sequence from nucleotide .876 to nucleotide 888. The untranslated region at the 3' end, excluding the consensus sequence, is 8 nucleotides shorter in the BRSV mRNA and, as seen in other RSV genes, has a lower homology (51%) with the HRSV counterpart region (Table 1) than the coding region. As in HRSV, there is no untranslated region, other the gene-start sequence, at the 5'-end of the M mRNA of BRSV. TABLE 1
Summary of the percentage of identity, at both the amino acid and nucleotide level, of the M protein and SH protein from BRSV and HRSV.
M protein
% Homology BRSVfA51908)/HRSVfA2)
Amino Acid 89
Nucleotide
Overall 74 - Coding region 80 3' end * 51
SH protein
% Homology
Figure imgf000026_0001
N-end (amino acids 1 to 23)
Hydrophobic domain (amino acids 24 to 41)
Ectodomain (amino acids 42 to 65)
* excluding the gene-start or gene-end consensus signals.
Intergenic region 24 % The intergenic region between M and SH genes of BRSV is 25 nucleotides long (vs 9 nucleotides in HRSV) and shares only 24% homology with the corresponding region in HRSV, suggesting that this region acts as a mere bridge between genes.
Finally, the polycistronic mRNA studied contains two additional open reading frames. One open reading frame from nucleotides 111 to 266 encodes a protein of 52 amino acids and overlaps with the M gene. A second open reading frame has also been reported for HRSV, encoding a protein of 75 amino acids, overlapping with the M gene (Satake and Venkateson, supra) . When the second open reading frames from BRSV and HRSV were compared no homology was found. Another open reading frame in BRSV M-SH polycistronic mRNA was found from nucleotides 1271 to 1423 which encodes a protein of 51 amino acids. No similar protein has been described for HRSV. Since these second open reading frames are not conserved, either at the sequence level or at the relative position in the genome, they probably do not play any role in the virus replication.
Other strains of BRSV can be grown and their genomes cloned in accordance with the description herein. The DNA sequences disclosed herein can be used as probes or to prepare degenerative primers for PCR to isolate the specific individual genes which can be cloned and utilized as described above for these additional strains of BRSV.
The present invention is further illustrated by reference to the following examples. These examples are provided for illustrative purposes, and are in no way intended to limit the scope of the invention. EXAMPLE 1 Isolation and Sequencing of BRSV Genes
The A51908 strain of BRSV originally isolated in Maryland and available from the American Type Culture Collection (ATCC VR-794) , was used in this Example. Madin-Darby bovine kidney (MDBK) cells were grow in Eagle's minimum essential medium with Earle's salts (MEM) containing 6% bovine fetal serum (BFS) . The BRSV strain A51908 was then prepared in MDBK cell cultures propagated in MEM containing 3% BFS. The viral mRNA was isolated from the BRSV-infected MDBK cells by the guanidinium thiocyanate-CsCl procedure (Chirgwin et al. , supra) , followed by two cycles of oligo (DT)-cellulose column chromotography. Double-stranded cDNA was synthesized by the RNase H method of Gubler and Hoffman, supra. The double-stranded cDNA molecules were ligated into the EcoRI site of plasmid Bluescript (Stratagene) . The resulting hybrid plasmids were used to transform E. coli JM109 cells using the method described by Cohen et al. , supra. Bacterial . clones containing recombinant DNA were selected on the basis of their color and resistance to ampicillin.
A total of 1642 ampicillin-resistant white colonies were obtained. All transformants were analyzed for the presence of viral sequences by colony hybridization with (1) 32P-labeled cDNAS made by reverse transcription of mRNAs extracted from BRSV-infection cells; and (2) 5'32P-labeled probe made from base hydrolyzed virion RNA. Over 60% of the total transfor ants (1016) hybridized strongly with both probes. The identity of these clones was determined by: (1) Northern blot analysis; and (2) in-vitro translation of hyrid selected mRNAS.
A cDNA clone was identified to contain sequences of the major nucleocapsid (N) gene of BRSV. This clone was used to identify other N gene clones by dot blot hydridization. One clone, designated A60, was selected for further analysis as it contained the largest insert among the N-specific clones. The nucleotide sequence of the mRNA of the N protein was obtained by sequencing the clone A60 by the dideoxynucleotide chain termination method. Clone A60 (1196 bp exclusive of poly(dA)) contained the entire N mRNA sequence lacking only 10 nucleotides of the 5' end of the mRNA. The nucleotide sequence was confirmed using clone A 1032 (1099 bp exclusive of poly (dA) ) , which expanded from nucleotide 97 to the poly (A) tail of the N mRNA.
The other BRSV genes of strain A51908 (the matrix protein gene, the phosphoprotein gene, the small hydrophobic protein gene, the fusion protein gene, the glycoprotein gene and the M2 protein gene) were isolated and sequenced using the same techniques. Furthermore, the phosphoprotein gene and the fusion protein gene of BRSV strain FS-l were also isolated and sequenced in the same manner. The sequences of the BRSV genes are set forth in the Sequence Listing as follows:
Strain A51908
SEQ ID NO:2 nucleocapsid protein (N) gene
SEQ ID NO:4 matrix protein (M) gene
SEQ ID NO:6 phosphoprotein (P) gene
SEQ ID NO:8 small hydrophobic (SH) protein gene
SEQ ID NO:10 fusion protein (F) gene
SEQ ID NO:12 glycoprotein (G) gene
SEQ ID NO:14 M2 protein (M2) gene
Strain FS-l
SEQ ID NO:16 phosphoprotein (P)
SEQ ID NO:18 fusion protein (F) gene EXAMPLE 2 Production of BRSV Genes With The Polymerase Chain Reaction
Two fragments of the BRSV N gene isolated in Example 1 are used as primers in the polymerase chain reaction (PCR) (U.S. Patent Nos. 4,683,195 and 4,683,202, both of which are incorporated herein by reference) . A DNA polymerase from Thermus aquaticus (U.S. Patent No. 4,889,818, incorporated herein by reference) is used in an amount of 1.25 units as the enzyme to catalyze the PCR.
In addition to the polymerase, 50 pmol of each primer, 10 copies of the BRSV genome, 200 μM of each dNTP, 2 mM MgCl2, 10 mM Tris-HCl (pH 8.3), and 50 mM KC1 are placed in a Perkin-Elmer Cetus Instruments Thermal Cycler. Thirty cycles of 96°C for 15 seconds, 50°C for 30 seconds and 75°C for 30 seconds are run. The BRSV N gene is then recovered using conventional techniques.
The other BRSV genes can also be produced using the above procedure.
EXAMPLE 3 Isolation of BRSV Proteins
The BRSV N gene isolated in Example 1 or produced in Example 2 is inserted into baculovirus (Autoqrapha California Nuclear Polyhedrosis Vrisu (AcNPV) (Voil et al. , J. Invertebrate Pathol. 22., 231 (1971)) using conventional techniques to form an expression vector. The expression vector is then used to infect Spodoptera frugiperda (SF-9) cells.
The SF-9 cells are maintained in a serum-free/protein-free medium developed by Maiorella et al. , Bio/Technology .6, 1406 (1988). The serum-free/ protein-free medium is composed of IPL/41 medium (JR Scientific, Woodland, CA) supplemented with tryptose phosphate broth (Oxoid USA, Columbia, MD) , fetal bovine serum (Gibco, Grand Island, NY) , and pluronic polyol F68 (BASF Wyandotte, Porsippamy, NJ) .
After infection with the baculovirus expression vector, the SF-9 cells are grown in a medium composed of IPL/41 medium supplemented with cod liver oil polyunsaturated fatty acid methyl esters, cholesterol, alpha-tocopherol acetate, Tween 80 and diluted Yestolate (Difco) . The cells are grown in spinner flasks, stirred at 75-100 rpm, at 27°C with an air atmosphere.
Approximately 4 to 5 days post-infection, the BRSV N protein titre peaks as cell lysis begins. SDS-PAGE analysis is then used to identify and isolate the recombinant BRSV N protein. The other BRSV genes from Example 1 may be used in the same procedure described above to isolate and identify their respective recombinant proteins.
While the invention has been described in connectin with specific embodiments thereof, it is understood that the invention is capable of further modifications. This disclosure is inteneded in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Samal, Siba K (ii) TITLE OF INVENTION: Bovine Respiratory Syncytial Virus Genes (iii) NUMBER OF SEQUENCES: 19
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Venable, Baetjer, Howard & Civiletti
(B) STREET: 1201 New York Avenue N.W. , suite 1000
(C) CITY: Washington
(D) STATE: DC
(E) COUNTRY: USA
(F) ZIP: 20005
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: WO
(B) FILING DATE: 04-NOV-1991
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/608,937
(B) FILING DATE: 05-NOV-1990
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Highet, David W
(B) REGISTRATION NUMBER: 30,265
(C) REFERENCE/DOCKET NUMBER: 20509-96711
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 202-962-4854
(B) TELEFAX: 202-962-8300
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7323 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI- SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus
(B) STRAIN: A 51908
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 1..1200
(D) OTHER INFORMATION: /label- N gene
(ix) FEATURE:
(A) NAME/KEY: π_isc_RNA
(B) LOCATION: 1204..2068
(D) OTHER INFORMATION: /label- P gene
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 2071..3019
(D) OTHER INFORMATION: /label- M gene
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 3045..3511
(D) OTHER INFORMATION: /label- SH gene
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 3549..4388
(D) OTHER INFORMATION: /label- G gene
(ix) FEATURE:
(A) NAME KEY: misc_RNA
(B) LOCATION: 4416..6309
(D) OTHER INFORMATION: /label- F gene
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 6363..7323
(D) OTHER INFORMATION: /label- M2 gene
(ix) FEATURE:
(A) NAME/KEY: misc_RNA
(B) LOCATION: 7256..7323
(D) OTHER INFORMATION: /label- 3' end L gene
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
GGGGCAAATA CAAAAATGGC TCTTAGCAAG GTCAAACTAA ATGACACTTT CAACAAGGAT 60
CAACTGTTAT CAACCAGCAA ATATACTATT CAACGTAGTA CAGGTGACAA CATTGATATA 120
CCCAATTATG ATGTACAAAA ACATCTCAAT AAGTTGTGTG GTATGCTACT AATAACAGAA 180
GATGCCAATC ATAAATTTAC AGGATTGATA GGTATATTAT ATGCTATGTC CCGATTGGGG 240 AGAGAAGATA CCCTTAAAAT ACTCAAAGAT GCAGGCTACC AAGTAAGGGC CAATGGGGTT 300
GATGTGATAA CACATCGACA GGATGTGAAT GGAAAAGAAA TGAAATTTGA AGTGCTAACA 360
TTAGTCAGCT TAACATCAGA AGTTCAAGGC AATATAGAAA TAGAGTCAAG GAAGTCTTAC 420
AAAAAGATGC TAAAAGAGAT GGGAGAGGTA GCGCCAGAAT ACAGACATGA CTCTCCTGAT 480
TGTGGTATGA TAGTGCTATG TGTTGCTGCT TTGGTTATAA CAAAATTAGC AGCAGGTGAT 540
AGATCAGGCC TCACTGCAGT CATTAGGAGA GCCAACAATG TACTAAGGAA TGAAATGAAA 600
CGATACAAAG GACTTATCCC GAAAGATATA GCTAACAGCT TCTATGAAGT GATTGAAAAG 660
TACCCTCATT ACATAGATGT ATTCGTACAT TTTGGCATTG CTCAATCCTC AACTAGAGGA 720
GGTAGTAGGG TAGAAGGAAT CTTTGCAGGG TTATTCATGA ATGCATATGG AGCAGGTCAA 780
GTGATGTTAA GATGGGGTGT ATTAGCCAAA TCAGTCAAGA ACATTATGCT TGGTCATGCC 840
AGCGTGCAAG CAGAAATGGA ACAGGTTGTA GAGGTCTATG AATATGCACA AAAGTTAGGT 900
GGAGAAGCTG GTTTTTATCA CATATTGAAC AACCCTAAAG CATCACTGTT ATCCTTAACA 960
CAATTCCCCA ACTTCTCTAG TGTAGTCCTA GGCAATGCTG CAGGACTAGG TATAATGGGT 1020
GAGTATAGAG GTACACCAAG AAACCAAGAC TTGTATGATG CTGCCAAAGC ATATGCGGAA 1080
CAATTAAAAG AGAATGGGGT CATCAATTAC AGTGTATTAG ATCTGACTAC AGAGGAACTA 1140
GAGGCAATCA AGAACCAATT GAATCCCAAA GACAATGATG TGGAACTGTG AGTAATAAAA 1200
CATGGGGCAA ATACGTCAGT ATGGAAAAAT TTGCACCTGA GTTTCATGGA GAAGATGCCA 1260
ATACAAAAGC AACCAAGTTT CTTGAATCCC TAAAAGGAAA ATTTACCTCT TCCAAGGATT 1320
CTAGGAAAAA AGATAGTATA ATATCAGTTA ATTCCATAGA CATAGAATTA CCTAAAGAAA 1380
GTCCTATAAC ATCTACCAAT CATAATATCA ACCAACCAAG TGAGATCAAT GACACTATTG 1440
CTGCAAATCA AGTTCATATC AGAAAGCCTT TGGTAAGCTT CAAAGAAGAA CTGCCATCAA 1500
GTGAAAACCC CTTTACAAAG CTGTATAAGG AAACTATAGA AACATTTGAC AATAATGAAG 1560
AAGAATCAAG CTACTCATAT GATGAGATAA ATGATCAAAC AAATGATAAT ATAACAGCAA 1620
GATTAGATAG GATAGATGAA AAATTAAGCG AGATAATAGG AATGCTCCAT ACACTAGTTG 1680
TGGCTAGTGC AGGACCAACA GCTGCTCGTG ACGGTATAAG AGATGCAATG GTAGGGCTCC 1740
GAGAAGAGAT GATTGAAAAA ATAAGATCAG AAGCTTTAAT GACTAACGAT AGATTAGAAG 1800
CAATGGCCAG GCTTAGGGAT GAAGAGAGTG AAAAGATGAC AAAAGATACA TCAGATGAAG 1860
TAAAATTAAC CCCTACCTCA GAGAAACTGA ACATGGTATT AGAAGATGAA AGTAGTGACA 1920 ATGATCTATC ACTTGAAGAT TTCTGAACAG CAACCAGCAC ACCAACCAAC AGATTGGTCA 1980
GATAGAACAA CCATCAATGA GAAAGCCAAC CGATCAGCCA GCCAACCAGT CACTCAACCA 2040
GCCTGTGATT CCACATAGTT AGTAAAAATT GGGGCAAATA TGGAGACATA CGTGAACAAA 2100
CTCCATGAAG GATCAATATA CACAGCTGCT GTTCAGTACA ATGTCATAGA GAAAGATGAT 2160
GATCCTGCAT CTCTCACAAT ATGGGTTCCC ATGTTCCAAT CATCCATCTC TGCCGATATG 2220
CTCATAAAAG AACTAATCAA TGTGAACATA TTAGTTCGAC AAATTTCTAC TCCGAAAGGT 2280
CCTTCATTGA AGATTATGAT AAACTCAAGA AGTGCTGTAC TAGCCCAAAT GCCCAGTAAA 2340
TTTACCATAA GTGCAAATGT ATCATTGGAT GAACGAAGTA AATTAGCCTA TGACATAACT 2400
ACTCCTTGTG AAATTAAGGC TTGTAGTTTA ACATGTTTAA AGGTGAAAAA TATGCTCACT 2460
ACTGTGAAAG ATCTCACCAT GAAAACATTC AATCCTACCC ATGAGATCAT TGCACTGTGT 2520
GAATTTGAAA ATATCATGAC ATCCAAAAGA GTTGTTATAC CAACTTTCTT AAGGTCAATC 2580
AATGTAAAAG CAAAGGATTT GGACTCACTA GAGAATATAG CTACCACAGA GTTTAAAAAT 2640
GCCATCACTA ATGCCAAAAT TATACCTTAT GCTGGGTTGG TGTTAGTTAT CACTGTAACT 2700
GACAATAAAG GGGCATTCAA GTACATTAAA CCACAAAGTC AATTTATAGT AGATCTTGGT 2760
GCATATCTAG AGAAGGAGAG CATATATTAT GTAACTACAA ATTGGAAACA CACAGCCACT 2820
AAATTCTCCA TTAAGCCTAT AGAGGACTGA TTCTCACACA ACTTATCTTA ACACAACAGA 2880
AGACTCCCTT GATAACTTAC AAATCATCAT GTGATCAAAT CCTATTTGCT GCTCTACCAA 2940
CCATAACCAT TATATGTTCT CAACCTGATC AACCCATCAA TTCATCTTGT AGATTATACC 3000
TCAATTAGAT AAATAAAAAT TATGAAAGTC AATAAAGATT ATGTGGGGTA AATAAAATCT 3060
GCATCCAATC AAGCACAGCA CACACCGGAC ACTCCTTGAA TCCACCAGCT GGTTGAACTT 3120
ATTACAATGA ACAATACATC TACCATGATA GAGTTTACTG GTAAATTTTG GACTTACTTT 3180
ACATTAGTCT TTATGATGTT AATCATAGGT TTTTTCTTTG TTATCACATC ACTAGTGGCG 3240
GCAATACTAA ACAAGTTATG TGACCTCAAC GATCATCATA CAAATAGTCT AGACATCAGA 3300
ACAGGGCTTA GGAATGATAC ACAATCAATA ACAAGAGCAC ATGTATGATC CATCAACCAA 3360
TCAAGCAAAA AAGAAGACAA CAAAACAAAA GAAAATAGCA ACATGCATCA AAGTTAAGCA 3420
AAGAAAAACC ACAATGGAAC AGACAGTCAT CACTCATATC CCTTTAGTCT ACAAATGCTG 3480
CATTATGTAC TGTTAATTAG TTATTTAAAA ATTACCTTAA AAATGGTTTA TGGTTACATA 3540
CAGATGTTGG GGCAAATACA AGCATGTCCA ACCACACCCA TCATCCTAAA TTCAAGACAT 3600 TAAAGAGGGC TTGGAAAGCC TCAAAATACT TCATAGTAGG ATTATCATGT TTATATAAGT 3660
TCAATTTAAA GTCCCTTGTC CAAACGGCTT TGACCTCCTT AGCAATGATA ACCTTGACAT 3720
CACTCGTCAT AACAGCCATT ATTTACATTA GTGTGGGAAA TGCTAAAGCC AAGCCCACAT 3780
CCAAACCAAC CACCCAACAA ACACAACAGC CCCAAAACCA CACCCCACTA CTTCCCACAG 3840
AGCACAACCA CAAATCAACT CACACATCAA CCCAAAGCAC CACACTGTCC CAACCAGCAA 3900
ACATAGACAC CACTAGTGGA ACTACATACG GTCACCCAAT CAACAGAACC CAAAACAGAA 3960
AAATCAAAAG CCAATCTACT CCACTTGCCA CCCGAAAACT ACCAATCAAC CCACTGGAAA 4020
GCAACCCCCC CGAAAACCAC CAAGACCACA ACAACTCCCA AACACTCCCT CATGTGCCCT 4080
GCAGCACATG CGAAGGCAAT CCTGCCTGTT CACCACTCTG CCAAATCGGG CTGGAGAGAG 4140
CACCAAGCAG AGCTCCCACA ATCACCCTCA AAAAGGCTCC AAAACCCAAA ACCACCAAAA 4200
AACCAACCAA GACAACAATC TACCACAGAA CCAGCCCTGA AGCCAAACTG CAAACCAAAA 4260
AAAACACGGC AACTCCACAA CAAGGCATCC TCTCTTCACC AGAACACCAA ACAAATCAAT 4320
CTACTACACA GATCTCACAA CACACCTCCA TATAATATCA ATTATGTTCA TATGTAGTTA 4380
TTTAAAAAGA TATGTATAAT TCACTAATTT AAACTGGGGC AAATAAGGAT GGCGACAACA 4440
ACCATGAGGA TGATCATCAG CATTATCCTC ATCTCTACCT ATGTGCCACA TATCACTTTA 4500
TGCCAGAACA TAACAGAAGA ATTTTATCAA TCGACATGCA GTGCAGTTAG TAGAGGTTAC 4560
CTTAGTGCAT TAAGAACTGG ATGGTATACA AGTGTGGTAA CAATAGAGTT GAGCAAAATA 4620
CAAAAAAATG TATGTAACGG TACTGATTCA AAAGTGAAAT TAATAAAGCA AGAACTAGAA 4680
AGATACAACA ATGCAGTAGC GGAATTGCAA TCACTTATGC AAAATGAACC GACCTCCTCT 4740
AGTAGAGCAA AAAGAGGGAT ACCAGAGTCG ATACATTATA CAAGAAACTC TACAAAAAAG 4800
TTTTATGGAC TAATGGGCAA AAAGAGAAAA AGGAGATTTT TAGGATTCTT GCTAGGTATT 4860
GGATCTGCTA TTGCAAGTGG TGTAGCAGTG TCCAAAGTAC TACACTTGGA GGGAGAGGTG 4920
AACAAAATTA AAAATGCACT GCTATCCACA AATAAAGCAG TGGTTAGTCT GTCCAATGGA 4980
GTTAGTGTCC TTACTAGCAA AGTACTTGAT CTAAAGAACT ATATAGACAA AGAGCTTCTA 5040
CCTAAAGTTA ACAATCATGA TTGTAGGATA TCCAACATAG CAACTGTGAT AGAATTCCAA 5100
CAAAAAAACA ATAGATTGTT GGAAATTGCT AGGGAATTTA GTGTAAATGC TGGTATTACC 5160
ACACCCCTCA GTACATACAT GTTAACCAAT AGTGAGTTAC TATCAATAAT TAATGATATG 5220
CCTATAACGA ATGACCAAAA AAAGCTAATG TCAGTATGTC AAATAGTCAG GCAACAGAGT 5280 TATTCCATCA TGTCAGTGTT AAGAGAGGTC ATAGCTTATG TTGTACAATT GCCTCTTTAT 5340
GGAGTTATAG ACACCCCCTG TTGGAAACTA CACACCTCTC CATTATGCAC CACTGATAAT 5400
AAAGAAGGGT CAAACATCTG CTTAACTAGG ACAGATCGTG GGTGGTATTG TGATAATGCA 5460
GGCTCTGTGT CTTTTTTCCC ACAAGCAGAG ACGTGTAAGG TACAATCAAA CAGAGTGTTC 5520
*
TGTGACACAA TGAACAGTTT AACTTTGCCT ACTGATGTTA ACTTATGCAA CACTGACATA 5580
TTCAATTCAA AGTATGATTG TAAAATAATG ACATCTAAAA CTGACATAAG TAGCTCTGTG 5640
ATAACTTCAA TAGGAGCTAT TGTATCATGC TATGGGAAGA CAAAATGTAC AGCCTCTAAT 5700
AAAAATCGTG GAATCATAAA GACTTTTTCC AATGGGTGTG ATTATGTATC AAACAAAGGA 5760
GTTGATACTG TATCAGTTGG TAACACACTA TATTATGTAA ATAAACTAGA GGGGAAAGCA 5820
CTCTATATAA AGGGTGAACC AATTATTAAT TACTATAATC CACTAGTATT TCCTTCTGAT 5880
GAGTTTGATG CATCAATTGC TCAAGTAAAC GCAAAAATAA ACCAAAGCCT GGCTTTCATA 5940
CGTCGATCTG ATGAGTTACT TCACAGTGTA GATGTAGGAA AATCCACCAC AAATGTAGTA 6000
ATTACTACTA TTATCATAGT GATAGTTGTA GTGATATTAA TGTTAATAAC TGTGGGATTA 6060
CTGTTTTACT GTAAGACCAG GAGTACACCT ATCATGTTAG GGAAGGATCA GCTTAGTAGT 6120
ATCAACAATC TTTCCTTTAG TAAATGAAAT GCATAATGTT TACAATCTTA ACCTCAGAAT 6180
CATAAATGTG ATGAGCCAAA TTTACTGATA CATTCAAAAG TTCCATCTGC CAAGACCTGC 6240
ATCTTTATCA GGTCTGCACA AGCTAACCTT ACATTCTATA CTCAGCTCTA TGTTAATAGT 6300
TATATAAAAG TATTATATTA ATCTCAAGAT CACCTATTTA ATAACCAATC ATTCAAAAAG 6360
ATGGGGCAAA TATGTCACGA AGAAATCCCT GCAAATATGA GATTAGGGGA CATTGCTTAA 6420
ATGGTAAAAA ATGCCATTTT AGTCATAATT ACTTTGAATG GCCTCCACAT GCTTTATTAG 6480
TGAGGCAAAA TTTTATGCTA AATAAGATAT TAAAATCTAT GGACAGGAAC AACGATACCC 6540
TGTCAGAAAT AAGTGGTGCT GCAGAATTAG ATAGAACAGA AGAATATGCA TTGGGTGTGA 6600
TAGGAGTTTT GGAAAGTTAC TTAAGCTCTA TCAATAATAT AACAAAACAA TCAGCCTGTG 6660
TTGCTATGAG TAAACTATTA GCCGAGATTA ACAATGATGA CATCAAGAGA TTAAGGAACA 6720
AGGAAGTGCC AACATCACCC AAGATAAGAA TATATAACAC AGTTATATCA TATATTGATA 6780
GCAACAAGAG AAACACAAAA CAAACTATAC ATTTGCTTAA GAGATTGCCT GCAGACGTGC 6840
TTAAAAAGAC CATCAAGAAC ACAATAGATA TTCACAACGA AATAAATGGT AATAACCAAG 6900
GTGACATAAA TGTTGATGAA CAAAATGAAT AACTCCAACA TTATTATTTT CCCAGAAAAA 6960 TACCCCTGTA GCATATCCTC TTTGCTAATT AAGGATGAAA ATGATGTTTT TGTACTAAGT 7020 CATCAGAATG TTCTTGACTG CTTACAGTTT CAATATCCAT ATAATATGTA TTCTCAAAAT 7080 CATATGCTTG ATGATATCTA TTGGACATCA CAGGAGCTGA TTGAGGATGT ACTTAAGATT 7140 CTTCATCTTT CTGGCATATC CATAAATAAG TATGTGATAT ATGTTTTAGT GCTATAGTAT 7200 ATAAGTCACT CAACTATTGA TCAACAGCCA CTTCTTCATA GCTAGCAATA TATAAGGACA 7260 AAATGGATAC ACTCATTCAT GAGAACTCAA CTAATGTTTA CTTAACAGAT AGTTATTTAA 7320 AAA 7323
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1197 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus
(B) STRAIN: A 51908
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 16..1188
(D) OTHER INFORMATION: /label- N gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGGGCAAATA CAAAA ATG GCT CTT AGC AAG GTC AAA CTA AAT GAC ACT TTC 51 Met Ala Leu Ser Lys Val Lys Leu Asn Asp Thr Phe 1 5 10
AAC AAG GAT CAA CTG TTA TCA ACC AGC AAA TAT ACT ATT CAA CGT AGT 99 Asn Lys Asp Gin Leu Leu Ser Thr Ser Lys Tyr Thr lie Gin Arg Ser 15 20 25
ACA GGT GAC AAC ATT GAT ATA CCC AAT TAT GAT GTA CAA AAA CAT CTC 147 Thr Gly Asp Asn lie Asp lie Pro Asn Tyr Asp Val Gin Lys His Leu 30 35 40 AAT AAG TTG TGT GGT ATG CTA CTA ATA ACA GAA GAT GCC AAT CAT AAA 195 Asn Lys Leu Cys Gly Met Leu Leu He Thr Glu Asp Ala Asn His Lys 45 50 55 60
TTT ACA GGA TTG ATA GGT ATA TTA TAT GCT ATG TCC CGA TTG GGG AGA 243 Phe Thr Gly Leu He Gly He Leu Tyr Ala Met Ser Arg Leu Gly Arg 65 70 75
GAA GAT ACC CTT AAA ATA CTC AAA GAT GCA GGC TAC CAA GTA AGG GCC 291 Glu Asp Thr Leu Lys He Leu Lys Asp Ala Gly Tyr Gin Val Arg Ala 80 85 90
AAT GGG GTT GAT GTG ATA ACA CAT CGA CAG GAT GTG AAT GGA AAA GAA 339 Asn Gly Val Asp Val He Thr His Arg Gin Asp Val Asn Gly Lys Glu 95 100 105
ATG AAA TTT GAA GTG CTA ACA TTA GTC AGC TTA ACA TCA GAA GTT CAA 387 Met Lys Phe Glu Val Leu Thr Leu Val Ser Leu Thr Ser Glu Val Gin 110 115 120
GGC AAT ATA GAA ATA GAG TCA AGG AAG TCT TAC AAA AAG ATG CTA AAA 435 Gly Asn He Glu He Glu Ser Arg Lys Ser Tyr Lys Lys Met Leu Lys 125 130 135 140
GAG ATG GGA GAG GTA GCC CCA GAA TAC AGA CAT GAC TCT CCT GAT TGT 483 Glu Met Gly Glu Val Ala Pro Glu Tyr Arg His Asp Ser Pro Asp Cys 145 150 155
GGT ATG ATA GTG CTA TGT GTT GCT GCT TTG GTT ATA ACA AAA TTA GCA 531 Gly Met He Val Leu Cys Val Ala Ala Leu Val He Thr Lys Leu Ala 160 165 170
GCA GGT GAT AGA TCA GGC CTC ACT GCA GTC ATT AGG AGA GCC AAC AAT 579 Ala Gly Asp Arg Ser Gly Leu Thr Ala Val He Arg Arg Ala Asn Asn 175 180 185
GTA CTA AGG AAT GAA ATG AAA CAA TAC AAA GGA CTT ATC CCG AAA GAT 627 Val Leu Arg Asn Glu Met Lys Gin Tyr Lys Gly Leu He Pro Lys Asp 190 195 200
ATA GCT AAC AGC TTC TAT GAA GTG ATT GAA AAG TAC CCT CAT TAC ATA 675 He Ala Asn Ser Phe Tyr Glu Val He Glu Lys Tyr Pro His Tyr He 205 210 215 220
GAT GTA TTC GTA CAT TTT GGC ATT GCT CAA TCC TCA ACT AGA GGA GGT 723 Asp Val Phe Val His Phe Gly He Ala Gin Ser Ser Thr Arg Gly Gly 225 230 235
AGT AGG GTA GAA GGA ATC TTT GCA GGG TTA TTC ATG AAT GCA TAT GGA 771 Ser Arg Val Glu Gly He Phe Ala Gly Leu Phe Met Asn Ala Tyr Gly 240 245 250
GCA GGT CAA GTG ATG TTA AGA TGG GGT GTA TTA GCC AAA TCA GTC AAG 819 Ala Gly Gin Val Met Leu Arg Trp Gly Val Leu Ala Lys Ser Val Lys 255 260 265 AAC ATT ATG CTT GGT CAT GCC AGC GTG CAA GCA GAA ATG GAA CAG GTT 867 Asn He Met Leu Gly His Ala Ser Val Gin Ala Glu Met Glu Gin Val 270 275 280
GTA GAG GTC TAT GAA TAT GCA CAA AAG TTA GGT GGA GAA GCT GGT TTT 915 Val Glu Val Tyr Glu Tyr Ala Gin Lys Leu Gly Gly Glu Ala Gly Phe 285 290 295 300
TAT CAC ATA TTG AAC AAC CCT AAA GCA TCA CTG TTA TCC TTA ACA CAA 963 Tyr His He Leu Asn Asn Pro Lys Ala Ser Leu Leu Ser Leu Thr Gin 305 310 315
TTC CCC AAC TTC TCT AGT GTA GTC CTA GGC AAT GCT GCA GGA CTA GGT 1011 Phe Pro Asn Phe Ser Ser Val Val Leu Gly Asn Ala Ala Gly Leu Gly 320 325 330
ATA ATG GGT GAG TAT AGA GGT ACA CCA AGA AAC CAA GAC TTG TAT GAT 1059 He Met Gly Glu Tyr Arg Gly Thr Pro Arg Asn Gin Asp Leu Tyr Asp 335 340 345
GCT GCC AAA GCA TAT GCG GAA CAA TTA AAA GAG AAT GGG GTC ATC AAT 1107 Ala Ala Lys Ala Tyr Ala Glu Gin Leu Lys Glu Asn Gly Val He Asn 350 355 360
TAC AGT GTA TTA GAT CTG ACT ACA GAG GAA CTA GAG GCA ATC AAG AAC 1155 Tyr Ser Val Leu Asp Leu Thr Thr Glu Glu Leu Glu Ala He Lys Asn 365 370 375 380
CAA TTG AAT CCC AAA GAC AAT GAT GTG GAA CTG TGGTATAAA 1197 Gin Leu Asn Pro Lys Asp Asn Asp Val Glu Leu 385 390
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 391 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Ala Leu Ser Lys Val Lys Leu Asn Asp Thr Phe Asn Lys Asp Gin 1 5 10 15
Leu Leu Ser Thr Ser Lys Tyr Thr He Gin Arg Ser Thr Gly Asp Asn 20 25 30
He Asp He Pro Asn Tyr Asp Val Gin Lys His Leu Asn Lys Leu Cys 35 40 45
Gly Met Leu Leu He Thr Glu Asp Ala Asn His Lys Phe Thr Gly Leu 50 55 60 Ile Gly He Leu Tyr Ala Met Ser Arg Leu Gly Arg Glu Asp Thr Leu 65 70 75 80
Lys He Leu Lys Asp Ala Gly Tyr Gin Val Arg Ala Asn Gly Val Asp 85 90 95
Val He Thr His Arg Gin Asp Val Asn Gly Lys Glu Met Lys Phe Glu 100 105 110
Val Leu Thr Leu Val Ser Leu Thr Ser Glu Val Gin Gly Asn He Glu 115 120 125
He Glu Ser Arg Lys Ser Tyr Lys Lys Met Leu Lys Glu Met Gly Glu 130 135 140
Val Ala Pro Glu Tyr Arg His Asp Ser Pro Asp Cys Gly Met He Val 145 150 155 160
Leu Cys Val Ala Ala Leu Val He Thr Lys Leu Ala Ala Gly Asp Arg 165 170 175
Ser Gly Leu Thr Ala Val He Arg Arg Ala Asn Asn Val Leu Arg Asn 180 185 190
Glu Met Lys Gin Tyr Lys Gly Leu He Pro Lys Asp He Ala Asn Ser 195 200 205
Phe Tyr Glu Val He Glu Lys Tyr Pro His Tyr He Asp Val Phe Val 210 215 220
His Phe Gly He Ala Gin Ser Ser Thr Arg Gly Gly Ser Arg Val Glu 225 230 235 240
Gly He Phe Ala Gly Leu Phe Met Asn Ala Tyr Gly Ala Gly Gin Val 245 250 255
Met Leu Arg Trp Gly Val Leu Ala Lys Ser Val Lys Asn He Met Leu 260 265 270
Gly His Ala Ser Val Gin Ala Glu Met Glu Gin Val Val Glu Val Tyr 275 280 285
Glu Tyr Ala Gin Lys Leu Gly Gly Glu Ala Gly Phe Tyr His He Leu 290 295 300
Asn Asn Pro Lys Ala Ser Leu Leu Ser Leu Thr Gin Phe Pro Asn Phe 305 310 315 320
Ser Ser Val Val Leu Gly Asn Ala Ala Gly Leu Gly He Met Gly Glu 325 330 335
Tyr Arg Gly Thr Pro Arg Asn Gin Asp Leu Tyr Asp Ala Ala Lys Ala 340 345 350
Tyr Ala Glu Gin Leu Lys Glu Asn Gly Val He Asn Tyr Ser Val Leu 355 360 365 Asp Leu Thr Thr Glu Glu Leu Glu Ala He Lys Asn Gin Leu Asn Pro 370 375 380
Lys Asp Asn Asp Val Glu Leu 385 390
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 949 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus
(B) STRAIN: A 51908
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 10..777
(D) OTHER INFORMATION: /label- M gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GGGGCAAAT ATG GAG ACA TAC GTG AAC AAA CTC CAT GAA GGA TCA ATA 48 Met Glu Thr Tyr Val Asn Lys Leu His Glu Gly Ser He 1 5 10
TAC ACA GCT GCT GTT CAG TAC AAT GTC ATA GAG AAA GAT GAT GAT CCT 96 Tyr Thr Ala Ala Val Gin Tyr Asn Val He Glu Lys Asp Asp Asp Pro 15 20 25
GCA TCT CTC ACA ATA TGG GTT CCC ATG TTC CAA TCA TCC ATC TCT GCC 144 Ala Ser Leu Thr He Trp Val Pro Met Phe Gin Ser Ser He Ser Ala 30 35 40 45
GAT ATG CTC ATA AAA GAA CTA ATC AAT GTG AAC ATA TTA GTT CGA CAA 192 Asp Met Leu He Lys Glu Leu He Asn Val Asn He Leu Val Arg Gin 50 55 60
ATT TCT ACT CCG AAA GGT CCT TCA TTG AAG ATT ATG ATA AAC TCA AGA 240 He Ser Thr Pro Lys Gly Pro Ser Leu Lys He Met He Asn Ser Arg 65 70 75 AGT GCT GTA CTA GCC CAA ATG CCC AGT AAA TTT ACC ATA AGT GCA AAT 288 Ser Ala Val Leu Ala Gin Met Pro Ser Lys Phe Thr He Ser Ala Asn 80 85 90
GTA TCA TTG GAT GAA CGA AGT AAA TTA GCC TAT GAC ATA ACT ACT CCT 336 Val Ser Leu Asp Glu Arg Ser Lys Leu Ala Tyr Asp He Thr Thr Pro 95 100 105
TGT GAA ATT AAG GCT TGT AGT TTA ACA TGT TTA AAG GTG AAA AAT ATG 384 Cys Glu He Lys Ala Cys Ser Leu Thr Cys Leu Lys Val Lys Asn Met 110 115 120 125
CTC ACT ACT GTG AAA GAT CTC ACC ATG AAA ACA TTC AAT CCT ACC CAT 432 Leu Thr Thr Val Lys Asp Leu Thr Met Lys Thr Phe Asn Pro Thr His 130 135 140
GAG ATC ATT GCA CTG TGT GAA TTT GAA AAT ATC ATG ACA TCC AAA AGA 480 Glu He He Ala Leu Cys Glu Phe Glu Asn He Met Thr Ser Lys Arg 145 150 155
GTT GTT ATA CCA ACT TTC TTA AGG TCA ATC AAT GTA AAA GCA AAG GAT 528 Val Val He Pro Thr Phe Leu Arg Ser He Asn Val Lys Ala Lys Asp 160 165 170
TTG GAC TCA CTA GAG AAT ATA GCT ACC ACA GAG TTT AAA AAT GCC ATC 576 Leu Asp Ser Leu Glu Asn He Ala Thr Thr Glu Phe Lys Asn Ala He 175 180 185
ACT AAT GCC AAA ATT ATA CCT TAT GCT GGG TTG GTG TTA GTT ATC ACT 624 Thr Asn Ala Lys He He Pro Tyr Ala Gly Leu Val Leu Val He Thr 190 195 200 205
GTA ACT GAC AAT AAA GGG GCA TTC AAG TAC ATT AAA CCA CAA AGT CAA 672 Val Thr Asp Asn Lys Gly Ala Phe Lys Tyr He Lys Pro Gin Ser Gin 210 215 220
TTT ATA GTA GAT CTT GGT GCA TAT CTA GAG AAG GAG AGC ATA TAT TAT 720 Phe He Val Asp Leu Gly Ala Tyr Leu Glu Lys Glu Ser He Tyr Tyr 225 230 235
GTA ACT ACA AAT TGG AAA CAC ACA GCC ACT AAA TTC TCC ATT AAG CCT 768 Val Thr Thr Asn Trp Lys His Thr Ala Thr Lys Phe Ser He Lys Pro 240 245 250
ATA GAG GAC TGATTCTCAC ACAACTTATC TTAACACAAC AGAAGACTCC 817
He Glu Asp 255
CTTGATAACT TACAAATCAT CATGTGATCA AATCCTATTT GCTGCTCTAC CAACCATAAC 877
CATTATATGT TCTCAACCTG ATCAACCCAT CAATTCATCT TGTAGATTAT ACCTCAATTA 937
GATAAATAAA AA 949 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 256 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Glu Thr Tyr Val Asn Lys Leu His Glu Gly Ser He Tyr Thr Ala 1 5 10 15
Ala Val Gin Tyr Asn Val He Glu Lys Asp Asp Asp Pro Ala Ser Leu 20 25 30
Thr He Trp Val Pro Met Phe Gin Ser Ser He Ser Ala Asp Met Leu 35 40 45
He Lys Glu Leu He Asn Val Asn He Leu Val Arg Gin He Ser Thr 50 55 60
Pro Lys Gly Pro Ser Leu Lys He Met He Asn Ser Arg Ser Ala Val 65 70 75 80
Leu Ala Gin Met Pro Ser Lys Phe Thr He Ser Ala Asn Val Ser Leu 85 90 95
Asp Glu Arg Ser Lys Leu Ala Tyr Asp He Thr Thr Pro Cys Glu He 100 105 110
Lys Ala Cys Ser Leu Thr Cys Leu Lys Val Lys Asn Met Leu Thr Thr 115 120 125
Val Lys Asp Leu Thr Met Lys Thr Phe Asn Pro Thr His Glu He He 130 135 140
Ala Leu Cys Glu Phe Glu Asn He Met Thr Ser Lys Arg Val Val He 145 150 155 160
Pro Thr Phe Leu Arg Ser He Asn Val Lys Ala Lys Asp Leu Asp Ser 165 170 175
Leu Glu Asn He Ala Thr Thr Glu Phe Lys Asn Ala He Thr Asn Ala 180 185 190
Lys He He Pro Tyr Ala Gly Leu Val Leu Val He Thr Val Thr Asp 195 200 205
Asn Lys Gly Ala Phe Lys Tyr He Lys Pro Gin Ser Gin Phe He Val 210 215 220
Asp Leu Gly Ala Tyr Leu Glu Lys Glu Ser He Tyr Tyr Val Thr Thr 225 230 235 240 Asn Trp Lys His Thr Ala Thr Lys Phe Ser He Lys Pro He Glu Asp 245 250 255
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 867 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus
(B) STRAIN: A 51908
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 18..740
(D) OTHER INFORMATION: /label- P gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GGGGCAAATA CGTCAGT ATG GAA AAA TTT GCA CCT GAG TTT CAT GGA GAA' 50
Met Glu Lys Phe Ala Pro Glu Phe His Gly Glu 1 5 10
GAT GCC AAT ACA AAA GCA ACC AAG TTT CTT GAA TCC CTA AAA GGA AAA 98 Asp Ala Asn Thr Lys Ala Thr Lys Phe Leu Glu Ser Leu Lys Gly Lys 15 20 25
TTT ACC TCT TCC AAG GAT TCT AGG AAA AAA GAT AGT ATA ATA TCA GTT 146 Phe Thr Ser Ser Lys Asp Ser Arg Lys Lys Asp Ser He He Ser Val 30 35 40
AAT TCC ATA GAC ATA GAA TTA CCT AAA GAA AGT CCT ATA ACA TCT ACC 194 Asn Ser He Asp He Glu Leu Pro Lys Glu Ser Pro He Thr Ser Thr 45 50 55
AAT CAT AAT ATC AAC CAA CCA AGT GAG ATC AAT GAC ACT ATT GCT GCA 242 Asn His Asn He Asn Gin Pro Ser Glu He Asn Asp Thr He Ala Ala 60 65 70 75
AAT CAA GTT CAT ATC AGA AAG CCT TTG GTA AGC TTC AAA GAA GAA CTG 290 Asn Gin Val His He Arg Lys Pro Leu Val Ser Phe Lys Glu Glu Leu 80 85 90 CCA TCA AGT GAA AAC CCC TTT ACA AAG CTG TAT AAG GAA ACT ATA GAA 338 Pro Ser Ser Glu Asn Pro Phe Thr Lys Leu Tyr Lys Glu Thr He Glu 95 100 105
ACA TTT GAC AAT AAT GAA GAA GAA TCA AGC TAC TCA TAT GAT GAG ATA 386 Thr Phe Asp Asn Asn Glu Glu Glu Ser Ser Tyr Ser Tyr Asp Glu He 110 115 120
AAT GAT CAA ACA AAT GAT AAT ATA ACA GCA AGA TTA GAT AGG ATA GAT 434 Asn Asp Gin Thr Asn Asp Asn He Thr Ala Arg Leu Asp Arg He Asp 125 130 135
GAA AAA TTA AGC GAG ATA ATA GGA ATG CTC CAT ACA CTA GTT GTG GCT 482 Glu Lys Leu Ser Glu He He Gly Met Leu His Thr Leu Val Val Ala 140 145 150 155
AGT GCA GGA CCA ACA GCT GCT CGT GAC GGT ATA AGA GAT GCA ATG GTA 530 Ser Ala Gly Pro Thr Ala Ala Arg Asp Gly He Arg Asp Ala Met Val 160 165 170
GGG CTC CGA GAA GAG ATG ATT GAA AAA ATA AGA TCA GAA GCT TTA ATG 578 Gly Leu Arg Glu Glu Met He Glu Lys He Arg Ser Glu Ala Leu Met 175 180 185
ACT AAC GAT AGA TTA GAA GCA ATG GCC AGG CTT AGG GAT GAA GAG AGT 626 Thr Asn Asp Arg Leu Glu Ala Met Ala Arg Leu Arg Asp Glu Glu Ser 190 195 200
GAA AAG ATG ACA AAA GAT ACA TCA GAT GAA GTA AAA TTA ACC CCT ACC 674 Glu Lys Met Thr Lys Asp Thr Ser Asp Glu Val Lys Leu Thr Pro Thr 205 210 215
TCA GAG AAA CTG AAC ATG GTA TTA GAA GAT GAA AGT AGT GAC AAT GAT 722 Ser Glu Lys Leu Asn Met Val Leu Glu Asp Glu Ser Ser Asp Asn Asp 220 225 230 235
CTA TCA CTT GAA GAT TTC TGAACAGCAA CCAGCACACC AACCAACAGA 770
Leu Ser Leu Glu Asp Phe 240
TTGGTCAGAT AGAACAACCA TCAATGAGAA AGCCAACCGA TCAGCCAGCC AACCAGTCAC 830
TCAACCAGCC TGTGATTCCA CATAGTTAGT AAAAAAA 867
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 241 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Met Glu Lys Phe Ala Pro Glu Phe His Gly Glu Asp Ala Asn Thr Lys 1 5 10 15
Ala Thr Lys Phe Leu Glu Ser Leu Lys Gly Lys Phe Thr Ser Ser Lys 20 25 30
Asp Ser Arg Lys Lys Asp Ser He He Ser Val Asn Ser He Asp He 35 40 45
Glu Leu Pro Lys Glu Ser Pro He Thr Ser Thr Asn His Asn He Asn 50 55 60
Gin Pro Ser Glu He Asn Asp Thr He Ala Ala Asn Gin Val His He 65 70 75 80
Arg Lys Pro Leu Val Ser Phe Lys Glu Glu Leu Pro Ser Ser Glu Asn 85 90 95
Pro Phe Thr Lys Leu Tyr Lys Glu Thr He Glu Thr Phe Asp Asn Asn 100 105 110
Glu Glu Glu Ser Ser Tyr Ser Tyr Asp Glu He Asn Asp Gin Thr Asn 115 120 125
Asp Asn He Thr Ala Arg Leu Asp Arg He Asp Glu Lys Leu Ser Glu 130 135 140
He He Gly Met Leu His Thr Leu Val Val Ala Ser Ala Gly Pro Thr 145 150 155 160
Ala Ala Arg Asp Gly He Arg Asp Ala Met Val Gly Leu Arg Glu Glu 165 170 175
Met He Glu Lys He Arg Ser Glu Ala Leu Met Thr Asn Asp Arg Leu 180 185 190
Glu Ala Met Ala Arg Leu Arg Asp Glu Glu Ser Glu Lys Met Thr Lys 195 200 205
Asp Thr Ser Asp Glu Val Lys Leu Thr Pro Thr Ser Glu Lys Leu Asn 210 215 220
Met Val Leu Glu Asp Glu Ser Ser Asp Asn Asp Leu Ser Leu Glu Asp 225 230 235 240
Phe
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 468 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine resoiratory syncytial virus
(B) STRAIN: A 51908
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 84..302
(D) OTHER INFORMATION: /label- SH gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
TGGGGTAAAT AAAATCTGCA TCCAATCAAG CACAGCACAC ACCGGACACT CCTTGAATCC 60
ACCAGCTGGT TGAACTTATT ACA ATG AAC AAT ACA TCT ACC ATG ATA GAG 110
Met Asn Asn Thr Ser Thr Met He Glu 1 5
TTT ACT GGT AAA TTT TGG ACT TAC TTT ACA TTA GTC TTT ATG ATG TTA 158 Phe Thr Gly Lys Phe Trp Thr Tyr Phe Thr Leu Val Phe Met Met Leu 10 15 20 25
ATC ATA GGT TTT TTC TTT GTT ATC ACA TCA CTA GTG GCG GCA ATA CTA 206 He He Gly Phe Phe Phe Val He Thr Ser Leu Val Ala Ala He Leu 30 35 40
AAC AAG TTA TGT GAC CTC AAC GAT CAT CAT ACA AAT AGT CTA GAC ATC 254 Asn Lys Leu Cys Asp Leu Asn Asp His His Thr Asn Ser Leu Asp He 45 50 55
AGA ACA GGG CTT AGG AAT GAT ACA CAA TCA ATA ACA AGA GCA CAT GTA 302 Arg Thr Gly Leu Arg Asn Asp Thr Gin Ser He Thr Arg Ala His Val 60 65 70
TGATCCATCA ACCAATCAAG CAAAAAAGAA GACAACAAAA CAAAAGAAAA TAGCAACATG 362
CATCAAAGTT AAGCAAAGAA AAACCACAAT GGAACAGACA GTCATCACTC ATATCCCTTT 422
AGTCTACAAA TGCTGCATTA TGTACTGTTA ATTAGTTATT TAAAAA 468
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 73 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Asn Asn Thr Ser Thr Met He Glu Phe Thr Gly Lys Phe Trp Thr 1 5 10 15
Tyr Phe Thr Leu Val Phe Met Met Leu He He Gly Phe Phe Phe Val 20 25 30
He Thr Ser Leu Val Ala Ala He Leu Asn Lys Leu Cys Asp Leu Asn 35 40 45
Asp His His Thr Asn Ser Leu Asp He Arg Thr Gly Leu Arg Asn Asp 50 55 60
Thr Gin Ser He Thr Arg Ala His Val 65 70
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1894 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus
(B) STRAIN: A 51908
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 14..1729
(D) OTHER INFORMATION: /label- F gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGGGCAAATA AGG ATG GCG ACA ACA ACC ATG AGG ATG ATC ATC AGC ATT 49 Met Ala Thr Thr Thr Met Arg Met He He Ser He 1 5 10
ATC CTC ATC TCT ACC TAT GTG CCA CAT ATC ACT TTA TGC CAG AAC ATA 97 He Leu He Ser Thr Tyr Val Pro His He Thr Leu Cys Gin Asn He 15 20 25 ACA GAA GAA TTT TAT CAA TCG ACA TGC AGT GCA GTT AGT AGA GGT TAC 145 Thr Glu Glu Phe Tyr Gin Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr 30 35 40
CTT AGT GCA TTA AGA ACT GGA TGG TAT ACA AGT GTG GTA ACA ATA GAG 193 Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val Thr He Glu 45 50 55 60
TTG AGC AAA ATA CAA AAA AAT GTA TGT AAC GGT ACT GAT TCA AAA GTG 241 Leu Ser Lys He Gin Lys Asn Val Cys Asn Gly Thr Asp Ser Lys Val 65 70 75
AAA TTA ATA AAG CAA GAA CTA GAA AGA TAC AAC AAT GCA GTA GCG GAA 289 Lys Leu He Lys Gin Glu Leu Glu Arg Tyr Asn Asn Ala Val Ala Glu 80 85 90
TTG CAA TCA CTT ATG CAA AAT GAA CCG ACC TCC TCT AGT AGA GCA AAA 337 Leu Gin Ser Leu Met Gin Asn Glu Pro Thr Ser Ser Ser Arg Ala Lys 95 100 105
AGA GGG ATA CCA GAG TCG ATA CAT TAT ACA AGA AAC TCT ACA AAA AAG 385 Arg Gly He Pro Glu Ser He His Tyr Thr Arg Asn Ser Thr Lys Lys 110 115 120
TTT TAT GGA CTA ATG GGC AAA AAG AGA AAA AGG AGA TTT TTA GGA TTC 433 Phe Tyr Gly Leu Met Gly Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe 125 130 135 140
TTG CTA GGT ATT GGA TCT GCT ATT GCA AGT GGT GTA GCA GTG TCC AAA 481 Leu Leu Gly He Gly Ser Ala He Ala Ser Gly Val Ala Val Ser Lys 145 150 155
GTA CTA CAC TTG GAG GGA GAG GTG AAC AAA ATT AAA AAT GCA CTG CTA 529 Val Leu His Leu Glu Gly Glu Val Asn Lys He Lys Asn Ala Leu Leu 160 165 170
TCC ACA AAT AAA GCA GTG GTT AGT CTG TCC AAT GGA GTT AGT GTC CTT 577 Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu 175 180 185
ACT AGC AAA GTA CTT GAT CTA AAG AAC TAT ATA GAC AAA GAG CTT CTA 625 Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr He Asp Lys Glu Leu Leu 190 195 200
CCT AAA GTT AAC AAT CAT GAT TGT AGG ATA TCC AAC ATA GCA ACT GTG 673 Pro Lys Val Asn Asn His Asp Cys Arg He Ser Asn He Ala Thr Val 205 210 215 220
ATA GAA TTC CAA CAA AAA AAC AAT AGA TTG TTG GAA ATT GCT AGG GAA 721 He Glu Phe Gin Gin Lys Asn Asn Arg Leu Leu Glu He Ala Arg Glu 225 230 235
TTT AGT GTA AAT GCT GGT ATT ACC ACA CCC CTC AGT ACA TAC ATG TTA 769 Phe Ser Val Asn Ala Gly He Thr Thr Pro Leu Ser Thr Tyr Met Leu 240 245 250 ACC AAT AGT GAG TTA CTA TCA ATA ATT AAT GAT ATG CCT ATA ACG AAT 817 Thr Asn Ser Glu Leu Leu Ser He He Asn Asp Met Pro He Thr Asn 255 260 265
GAC CAA AAA AAG CTA ATG TCA GTA TGT CAA ATA GTC AGG CAA CAG AGT 865 Asp Gin Lys Lys Leu Met Ser Val Cys Gin He Val Arg Gin Gin Ser 270 275 280
TAT TCC ATC ATG TCA GTG TTA AGA GAG GTC ATA GCT TAT GTT GTA CAA 913 Tyr Ser He Met Ser Val Leu Arg Glu Val He Ala Tyr Val Val Gin 285 290 295 300
TTG CCT CTT TAT GGA GTT ATA GAC ACC CCC TGT TGG AAA CTA CAC ACC 961 Leu Pro Leu Tyr Gly Val He Asp Thr Pro Cys Trp Lys Leu His Thr 305 310 315
TCT CCA TTA TGC ACC ACT GAT AAT AAA GAA GGG TCA AAC ATC TGC TTA 1009 Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn He Cys Leu 320 325 330
ACT AGG ACA GAT CGT GGG TGG TAT TGT GAT AAT GCA GGC TCT GTG TCT 1057 Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser 335 340 345
TTT TTC CCA CAA GCA GAG ACG TGT AAG GTA CAA TCA AAC AGA GTG TTC 1105 Phe Phe Pro Gin Ala Glu Thr Cys Lys Val Gin Ser Asn Arg Val Phe 350 355 360
TGT GAC ACA ATG AAC AGT TTA ACT TTG CCT ACT GAT GTT AAC TTA TGC 1153 Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys 365 370 375 380
AAC ACT GAC ATA TTC AAT TCA AAG TAT GAT TGT AAA ATA ATG ACA TCT 1201 Asn Thr Asp He Phe Asn Ser Lys Tyr Asp Cys Lys He Met Thr Ser 385 390 395
AAA ACT GAC ATA AGT AGC TCT GTG ATA ACT TCA ATA GGA GCT ATT GTA 1249 Lys Thr Asp He Ser Ser Ser Val He Thr Ser He Gly Ala He Val 400 405 410
TCA TGC TAT GGG AAG ACA AAA TGT ACA GCC TCT AAT AAA AAT CGT GGA 1297 Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly 415 420 425
ATC ATA AAG ACT TTT TCC AAT GGG TGT GAT TAT GTA TCA AAC AAA GGA 1345 He He Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly 430 435 440
GTT GAT ACT GTA TCA GTT GGT AAC ACA CTA TAT TAT GTA AAT AAA CTA 1393 Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu 445 450 455 460
GAG GGG AAA GCA CTC TAT ATA AAG GGT GAA CCA ATT ATT AAT TAC TAT 1441 Glu Gly Lys Ala Leu Tyr He Lys Gly Glu Pro He He Asn Tyr Tyr 465 470 475 AAT CCA CTA GTA TTT CCT TCT GAT GAG TTT GAT GCA TCA ATT GCT CAA 1489 Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser He Ala Gin 480 485 490
GTA AAC GCA AAA ATA AAC CAA AGC CTG GCT TTC ATA CGT CGA TCT GAT 1537 Val Asn Ala Lys He Asn Gin Ser Leu Ala Phe He Arg Arg Ser Asp 495 500 505
GAG TTA CTT CAC AGT GTA GAT GTA GGA AAA TCC ACC ACA AAT GTA GTA 1585 Glu Leu Leu His Ser Val Asp Val Gly Lys Ser Thr Thr Asn Val Val 510 515 520
ATT ACT ACT ATT ATC ATA GTG ATA GTT GTA GTG ATA TTA ATG TTA ATA 1633 He Thr Thr He He He Val He Val Val Val He Leu Met Leu He 525 530 535 540
ACT GTG GGA TTA CTG TTT TAC TGT AAG ACC AGG AGT ACA CCT ATC ATG 1681 Thr Val Gly Leu Leu Phe Tyr Cys Lys Thr Arg Ser Thr Pro He Met 545 550 555
TTA GGG AAG GAT CAG CTT AGT AGT ATC AAC AAT CTT TCC TTT AGT AAA 1729 Leu Gly Lys Asp Gin Leu Ser Ser He Asn Asn Leu Ser Phe Ser Lys 560 565 570
TGAAATGCAT AATGTTTACA ATCTTAACCT CAGAATCATA AATGTGATGA GCCAAATTTA 1789
CTGATACATT CAAAAGTTCC ATCTGCCAAG ACCTGCATCT TTATCAGGTC TGCACAAGCT 1849
AACCTTACAT TCTATACTCA GCTCTATGTT AATAGTTATA TAAAA 1894
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 572 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Ala Thr Thr Thr Met Arg Met He He Ser He He Leu He Ser 1 5 10 15
Thr Tyr Val Pro His He Thr Leu Cys Gin Asn He Thr Glu Glu Phe 20 25 30
Tyr Gin Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr He Glu Leu Ser Lys He 50 55 60
Gin Lys Asn Val Cys Asn Gly Thr Asp Ser Lys Val Lys Leu He Lys 65 70 75 80 Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Ala Glu Leu Gin Ser Leu 85 90 95
Met Gin Asn Glu Pro Thr Ser Ser Ser Arg Ala Lys Arg Gly He Pro 100 105 110
Glu Ser He His Tyr Thr Arg Asn Ser Thr Lys Lys Phe Tyr Gly Leu 115 120 125
Met Gly Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly He 130 135 140
Gly Ser Ala He Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu 145 150 155 160
Glu Gly Glu Val Asn Lys He Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175
Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190
Leu Asp Leu Lys Asn Tyr He Asp Lys Glu Leu Leu Pro Lys Val Ash 195 200 205
Asn His Asp Cys Arg He Ser Asn He Ala Thr Val He Glu Phe Gin 210 215 220
Gin Lys Asn Asn Arg Leu Leu Glu He Ala Arg Glu Phe Ser Val Asn 225 230 235 240
Ala Gly He Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255
Leu Leu Ser He He Asn Asp Met Pro He Thr Asn Asp Gin Lys Lys 260 265 270
Leu Met Ser Val Cys Gin He Val Arg Gin Gin Ser Tyr Ser He Met 275 280 285
Ser Val Leu Arg Glu Val He Ala Tyr Val Val Gin Leu Pro Leu Tyr 290 295 300
Gly Val He Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys 305 310 315 320
Thr Thr Asp Asn Lys Glu Gly Ser Asn He Cys Leu Thr Arg Thr Asp 325 330 335
Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gin 340 345 350
Ala Glu Thr Cys Lys Val Gin Ser Asn Arg Val Phe Cys Asp Thr Met 355 360 365
Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp He 370 375 380 Phe Asn Ser Lys Tyr Asp Cys Lys He Met Thr Ser Lys Thr Asp He 385 390 395 400
Ser Ser Ser Val He Thr Ser He Gly Ala He Val Ser Cys Tyr Gly 405 410 415
Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly He He Lys Thr 420 425 430
Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val 435 440 445
Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala 450 455 460
Leu Tyr He Lys Gly Glu Pro He He Asn Tyr Tyr Asn Pro Leu Val 465 470 475 480
Phe Pro Ser Asp Glu Phe Asp Ala Ser He Ala Gin Val Asn Ala Lys 485 490 495
He Asn Gin Ser Leu Ala Phe He Arg Arg Ser Asp Glu Leu Leu His 500 505 510
Ser Val Asp Val Gly Lys Ser Thr Thr Asn Val Val He Thr Thr He 515 520 525
He He Val He Val Val Val He Leu Met Leu He Thr Val Gly Leu 530 535 540
Leu Phe Tyr Cys Lys Thr Arg Ser Thr Pro He Met Leu Gly Lys Asp 545 550 555 560
Gin Leu Ser Ser He Asn Asn Leu Ser Phe Ser Lys 565 570
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 840 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus
(B) STRAIN: A51908 (ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 16..804
(D) OTHER INFORMATION: /label- G gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GGGGCAAATA CAAGC ATG TCC AAC CAC ACC CAT CAT CCT AAA TTC AAG ACA 51 Met Ser Asn His Thr His His Pro Lys Phe Lys Thr 1 5 10
TTA AAG AGG GCT TGG AAA GCC TCA AAA TAC TTC ATA GTA GGA TTA TCA 99 Leu Lys Arg Ala Trp Lys Ala Ser Lys Tyr Phe He Val Gly Leu Ser 15 20 25
TGT TTA TAT AAG TTC AAT TTA AAG TCC CTT GTC CAA ACG GCT TTG ACC 147 Cys Leu Tyr Lys Phe Asn Leu Lys Ser Leu Val Gin Thr Ala Leu Thr 30 35 40
TCC TTA GCA ATG ATA ACC TTG ACA TCA CTC GTC ATA ACA GCC ATT ATT 195 Ser Leu Ala Met He Thr Leu Thr Ser Leu Val He Thr Ala He He 45 50 55 60
TAC ATT AGT GTG GGA AAT GCT AAA GCC AAG CCC ACA TCC AAA CCA ACC 243 Tyr He Ser Val Gly Asn Ala Lys Ala Lys Pro Thr Ser Lys Pro Thr 65 70 75
ACC CAA CAA ACA CAA CAG CCC CAA AAC CAC ACC CCA CTA CTT CCC ACA 291 Thr Gin Gin Thr Gin Gin Pro Gin Asn His Thr Pro Leu Leu Pro Thr 80 85 90
GAG CAC AAC CAC AAA TCA ACT CAC ACA TCA ACC CAA AGC ACC ACA CTG 339 Glu His Asn His Lys Ser Thr His Thr Ser Thr Gin Ser Thr Thr Leu 95 100 105
TCC CAA CCA CCA AAC ATA GAC ACC ACT AGT GGA ACT ACA TAC GGT CAC 387 Ser Gin Pro Pro Asn He Asp Thr Thr Ser Gly Thr Thr Tyr Gly His 110 115 120
CCA ATC AAC AGA ACC CAA AAC AGA AAA ATC AAA AGC CAA TCT ACT CCA 435 Pro He Asn Arg Thr Gin Asn Arg Lys He Lys Ser Gin Ser Thr Pro 125 130 135 140
CTT GCC ACC CGA AAA CTA CCA ATC AAC CCA CTG GAA AGC AAC CCC CCC 483 Leu Ala Thr Arg Lys Leu Pro He Asn Pro Leu Glu Ser Asn Pro Pro 145 150 155
GAA AAC CAC CAA GAC CAC AAC AAC TCC CAA ACA CTC CCT CAT GTG CCC 531 Glu Asn His Gin Asp His Asn Asn Ser Gin Thr Leu Pro His Val Pro 160 165 170
TGC AGC ACA TGC GAA GGC AAT CCT GCC TGT TCA CCA CTC TGC CAA ATC 579 Cys Ser Thr Cys Glu Gly Asn Pro Ala Cys Ser Pro Leu Cys Gin He 175 180 185 GGG CTG GAG AGA GCA CCA AGC AGA GCT CCC ACA ATC ACC CTC AAA AAG 627 Gly Leu Glu Arg Ala Pro Ser Arg Ala Pro Thr He Thr Leu Lys Lys 190 195 200
GCT CCA AAA CCC AAA ACC ACC AAA AAA CCA ACC AAG ACA ACA ATC TAC 675 Ala Pro Lys Pro Lys Thr Thr Lys Lys Pro Thr Lys Thr Thr He Tyr 205 210 215 220
CAC AGA ACC AGC CCT GAA GCC AAA CTG CAA ACC AAA AAA AAC ACG GCA 723 His Arg Thr Ser Pro Glu Ala Lys Leu Gin Thr Lys Lys Asn Thr Ala 225 230 235
ACT CCA CAA CAA GGC ATC CTC TCT TCA CCA GAA CAC CAA ACA AAT CAA 771 Thr Pro Gin Gin Gly He Leu Ser Ser Pro Glu His Gin Thr Asn Gin 240 245 250
TCT ACT ACA CAG ATC TCA CAA CAC ACC TCC ATA TAATATCAAT TATGTTCATA 824 Ser Thr Thr Gin He Ser Gin His Thr Ser He 255 260
TGTAGTTATT TAAAAA 840
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 263 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Ser Asn His Thr His His Pro Lys Phe Lys Thr Leu Lys Arg Ala 1 5 10 15
Trp Lys Ala Ser Lys Tyr Phe He Val Gly Leu Ser Cys Leu Tyr Lys 20 25 30
Phe Asn Leu Lys Ser Leu Val Gin Thr Ala Leu Thr Ser Leu Ala Met 35 40 45
He Thr Leu Thr Ser Leu Val He Thr Ala He He Tyr He Ser Val 50 55 60
Gly Asn Ala Lys Ala Lys Pro Thr Ser Lys Pro Thr Thr Gin Gin Thr 65 70 75 80
Gin Gin Pro Gin Asn His Thr Pro Leu Leu Pro Thr Glu His Asn His 85 90 95
Lys Ser Thr His Thr Ser Thr Gin Ser Thr Thr Leu Ser Gin Pro Pro 100 105 110 Asn He Asp Thr Thr Ser Gly Thr Thr Tyr Gly His Pro He Asn Arg 115 120 125
Thr Gin Asn Arg Lys He Lys Ser Gin Ser Thr Pro Leu Ala Thr Arg 130 135 140
Lys Leu Pro He Asn Pro Leu Glu Ser Asn Pro Pro Glu Asn His Gin 145 150 155 160
Asp His Asn Asn Ser Gin Thr Leu Pro His Val Pro Cys Ser Thr Cys 165 170 175
Glu Gly Asn Pro Ala Cys Ser Pro Leu Cys Gin He Gly Leu Glu Arg 180 185 190
Ala Pro Ser Arg Ala Pro Thr He Thr Leu Lys Lys Ala Pro Lys Pro 195 200 205
Lys Thr Thr Lys Lys Pro Thr Lys Thr Thr He Tyr His Arg Thr Ser 210 215 220
Pro Glu Ala Lys Leu Gin Thr Lys Lys Asn Thr Ala Thr Pro Gin Gin 225 230 235 240
Gly He Leu Ser Ser Pro Glu His Gin Thr Asn Gin Ser Thr Thr Gin 245 250 255
He Ser Gin His Thr Ser He 260
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 961 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus
(B) STRAIN: A 51908
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 10..567
(D) OTHER INFORMATION: /label- M2 gene (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GGGGCAAAT ATG TCA CGA AGA AAT CCC TGC AAA TAT GAG ATT AGG GGA 48 Met Ser Arg Arg Asn Pro Cys Lys Tyr Glu He Arg Gly 1 5 10
CAT TGC TTA AAT GGT AAA AAA TGC CAT TTT AGT CAT AAT TAC TTT GAA 96 His Cys Leu Asn Gly Lys Lys Cys His Phe Ser His Asn Tyr Phe Glu 15 20 25
TGG CCT CCA CAT GCT TTA TTA GTG AGG CAA AAT TTT ATG CTA AAT AAG 144 Trp Pro Pro His Ala Leu Leu Val Arg Gin Asn Phe Met Leu Asn Lys 30 35 40 45
ATA TTA AAA TCT ATG GAC AGG AAC AAC GAT ACC CTG TCA GAA ATA AGT 192 He Leu Lys Ser Met Asp Arg Asn Asn Asp Thr Leu Ser Glu He Ser 50 55 60
GGT GCT GCA GAA TTA GAT AGA ACA GAA GAA TAT GCA TTG GGT GTG ATA 240 Gly Ala Ala Glu Leu Asp Arg Thr Glu Glu Tyr Ala Leu Gly Val He 65 70 75
GGA GTT TTG GAA AGT TAC TTA AGC TCT ATC AAT AAT ATA ACA AAA CAA 288 Gly Val Leu Glu Ser Tyr Leu Ser Ser He Asn Asn He Thr Lys Gin 80 85 90
TCA GCC TGT GTT GCT ATG AGT AAA CTA TTA GCC GAG ATT AAC AAT GAT 336 Ser Ala Cys Val Ala Met Ser Lys Leu Leu Ala Glu He Asn Asn Asp 95 100 105
GAC ATC AAG AGA TTA AGG AAC AAG GAA GTG CCA ACA TCA CCC AAG ATA 384 Asp He Lys Arg Leu Arg Asn Lys Glu Val Pro Thr Ser Pro Lys lie 110 115 120 125
AGA ATA TAT AAC ACA GTT ATA TCA TAT ATT GAT AGC AAC AAG AGA AAC 432 Arg He Tyr Asn Thr Val He Ser Tyr He Asp Ser Asn Lys Arg Asn 130 135 140
ACA AAA CAA ACT ATA CAT TTG CTT AAG AGA TTG CCT GCA GAC GTG CTT 480 Thr Lys Gin Thr He His Leu Leu Lys Arg Leu Pro Ala Asp Val Leu 145 150 155
AAA AAG ACC ATC AAG AAC ACA ATA GAT ATT CAC AAC GAA ATA AAT GGT 528 Lys Lys Thr He Lys Asn Thr He Asp He His Asn Glu He Asn Gly 160 165 170
AAT AAC CAA GGT GAC ATA AAT GTT GAT GAA CAA AAT GAA TAACTCCAAC 577 Asn Asn Gin Gly Asp He Asn Val Asp Glu Gin Asn Glu 175 180 185
ATTATTATTT TCCCAGAAAA ATACCCCTGT AGCATATCCT CTTTGCTAAT TAAGGATGAA 637
AATGATGTTT TTGTACTAAG TCATCAGAAT GTTCTTGACT GCTTACAGTT TCAATATCCA 697
TATAATATGT ATTCTCAAAA TCATATGCTT GATGATATCT ATTGGACATC ACAGGAGCTG 757 ATTGAGGATG TACTTAAGAT TCTTCATCTT TCTGGCATAT CCATAAATAA GTATGTGATA 817
TATGTTTTAG TGCTATAGTA TATAAGTCAC TCAACTATTG ATCAACAGCC ACTTCTTCAT 877
AGCTAGCAAT ATATAAGGAC AAAATGGATA CACTCATTCA TGAGAACTCA ACTAATGTTT 937
ACTTAACAGA TAGTTATTTA AAAA 961
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 186 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met Ser Arg Arg Asn Pro Cys Lys Tyr Glu He Arg Gly His Cys Leu 1 5 10 15
Asn Gly Lys Lys Cys His Phe Ser His Asn Tyr Phe Glu Trp Pro Pro 20 25 30
His Ala Leu Leu Val Arg Gin Asn Phe Met Leu Asn Lys He Leu Lys 35 40 45
Ser Met Asp Arg Asn Asn Asp Thr Leu Ser Glu He Ser Gly Ala Ala 50 55 60
Glu Leu Asp Arg Thr Glu Glu Tyr Ala Leu Gly Val He Gly Val Leu 65 70 75 80
Glu Ser Tyr Leu Ser Ser He Asn Asn He Thr Lys Gin Ser Ala Cys 85 90 95
Val Ala Met Ser Lys Leu Leu Ala Glu He Asn Asn Asp Asp He Lys 100 105 110
Arg Leu Arg Asn Lys Glu Val Pro Thr Ser Pro Lys He Arg He Tyr 115 120 125
Asn Thr Val He Ser Tyr He Asp Ser Asn Lys Arg Asn Thr Lys Gin 130 135 140
Thr He His Leu Leu Lys Arg Leu Pro Ala Asp Val Leu Lys Lys Thr 145 150 155 160
He Lys Asn Thr He Asp He His Asn Glu He Asn Gly Asn Asn Gin 165 170 175
Gly Asp He Asn Val Asp Glu Gin Asn Glu 180 185 (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 833 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus
(B) STRAIN: FS-l
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..709
(D) OTHER INFORMATION: /label- P gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GCCTGAGTTT CATGGAGAAG ATGCCAATAC AAAAGCAACC AAGTTTCTTG AATCCCTAAA 60
AGGGAAATTT ACTTCTTCTA AGGATTCTAG GAAAAAAGAT AGTATAATAT CAGTTAATTC 120
CATAGACATA GAATTACCTA AAGAGAGTCC TATAACATCT ACCAATCAAA ATATCAACCA 180
ACTAAGTGAG ATCAATGACA CTATTGCTAC GAATCAAGTT CATATCAGAA AGCCTTTGGT 240
AAGCTTCAAA GAAGAACTGC CATCAAGTGA AAACCCCTTT ACAAGGCTGT ATAAGGAAAC 300
TATAGAAACA TTTGACAATA ATGAAGAAGA ATCAAGCTAC TCATATGATG AGATAAATGA 360
TCAAACAAAT GATAATATAA CAGCAAGACT AGATAGGATA GATGAAAAAT TAAGCGAGAT 420
AATAGGAATG CTCCATACAT TAGTTGTGGC TAGTGCAGGA CCAACAGCTG CTCGTGACGG 480
TATAAGAGAT GCCATGGTAG GGCTCCGAGA AGAGATGATT GAGAAAATAA GATCAGAAGC 540
TTTAATGACT AACGATAGGT TAGAAGCAAT GGCCAGGCTT AGGAATGAAG AGAGTGAAAA 600
GATGAGAAAA GATACATCAG ATGAAGTAAA ATTAACCCCT ACCTCAGAGA AGCTGAACAT 660
GGTATTAGAA GATGAAAGTA GTGACAATGA TCTATCACTT GAAGATTTCT GAATAGCAAC 720
CAGCCCACCC ACCAACAGAT TGGTCAGATA GATCAACCAT CAATGATAAA GCCAACTAAT 780
CAACCAGCCA ACCAGTCACT CAACCAGCCT GTGATTCCAC ATAGTTAGTA AAA 833 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 236 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus
(B) STRAIN: FS-l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Pro Glu Phe His Gly Glu Asp Ala Asn Thr Lys Ala Thr Lys Phe Leu 1 5 10 15
Glu Ser Leu Lys Gly Lys Phe Thr Ser Ser Lys Asp Ser Arg Lys Lys 20 25 30
Asp Ser He He Ser Val Asn Ser He Asp He Glu Leu Pro Lys Glu 35 40 45
Ser Pro He Thr Ser Thr Asn Gin Asn He Asn Gin Leu Ser Glu He 50 55 60
Asn Asp Thr He Ala Thr Asn Gin Val His He Arg Lys Pro Leu Val 65 70 ' 75 80
Ser Phe Lys Glu Glu Leu Pro Ser Ser Glu Asn Pro Phe Thr Arg Leu 85 90 95
Tyr Lys Glu Thr He Glu Thr Phe Asp Asn Asn Glu Glu Glu Ser Ser 100 105 110
Tyr Ser Tyr Asp Glu He Asn Asp Gin Thr Asn Asp Asn He Thr Ala 115 120 125
Arg Leu Asp Arg He Asp Glu Lys Leu Ser Glu He He Gly Met Leu 130 135 140
His Thr Leu Val Val Ala Ser Ala Gly Pro Thr Ala Ala Arg Asp Gly 145 150 155 160
He Arg Asp Ala Met Val Gly Leu Arg Glu Glu Met He Glu Lys He 165 170 175
Arg Ser Glu Ala Leu Met Thr Asn Asp Arg Leu Glu Ala Met Ala Arg 180 185 190
Leu Arg Asn Glu Glu Ser Glu Lys Met Xaa Lys Asp Thr Ser Asp Glu 195 200 205 Val Lys Leu Thr Pro Thr Ser Glu Lys Leu Asn Met Val Leu Glu Asp 210 215 220
Glu Ser Ser Asp Asn Asp Leu Ser Leu Glu Asp Phe 225 230 235
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1879 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus
(B) STRAIN: FS-l
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 13..1728
(D) OTHER INFORMATION: /label- F gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GGGACAACAG CCATGAGGAT GGTCATCAGC ATTATCTTCA TCTCTACCTA TGTGACACAT 60
ATCACTTTAT GCCAAAACAT AACAGAAGAA TTTTATCAAT CAACATGCAG TGCAGTTAGT 120
AGAGGTTACC TTAGTGCATT AAGAACTGGA TGGTATACAA GTGTGGTAAC AATAGAGTTG 180
AGCAAAATAC AAAAAAATGT GTGTAAAAGT ACTGATTCAA AAGTGAAATT AATAAAGCAA 240
GAACTAGAAA GATACAACAA TGCAGTAATA GAATTGCAGT CACTTATGCA AAATGAACCG 300
GCCTCCTTCA GTAGAGCAAA AAGAGGGATA CCAGAGTTGA TACATTATCC AAGAAACTCT 360
ACAAAAAGGT TTTATGGGCT AATGGGCAAG AAGAGAAAAA GGAGACATTT TAGATTCTTG 420
CTAGGTATTG GATCTGCTAT TGCAAGTGGT GTAGCAGTGT CCAAAGTACT ACACCTGGAG 480
GGAGAGGTGA ATAAAATTAA AAATGCACTG CTATCCACAA ATAAAGCAGT AGTTAGTCTA 540
TCCAATGGAG TTAGTGTCCT TACTAGCAAA GTACTTGATC TAAAGAACTA TATAGACAAA 600
GAGCTTCTAC CTAAAGTTAA CAATCATGAT TGTAGGATAT CCAACATAGG AACTGTGATA 660 GAATTCCAAC AAAAAAACAA TAGATTGTTA GAAATTGCTA GGGAATTTAG TGTAAATGCT 720
GGTATTACCA CACCCCTCAG TACATACATG TTGACCAATA GTGAATTACT ATCACTAATT 780
AATGATATGC CTATAACGAA TGACCAAAAA AAGCTAATGT CAGTATGTCA AATAGTCAGG 840
CAACAGAGTT ATTCCATTAT GTCAGTGTCA AGAGAGGTCA TAGCTTATGA AGTACAATTG 900
CCTATTTATG GAGTTATAGA CACCCCCTGT TGGAAAATAC ACACCTCTCC ATTATGCACC 960
ACTGATAATA AAGAAGGGTC AAACATCTGC TTAACTAGGA CAGATCGTGG GTGGTATTGT 1020
GACAATGCAG GCTCTGTGTC TTTTTTCCCA CAGGCAGAGA CATGTAAGGT ACAATCAAAT 1080
AGAGTGTTCT GTGACACAAT GAACAGTTTA ACTCTGCCTA CTGATGTTAA CTTATGCAAC 1140
ACTGACATAT TCAATACAAA GTATGACTGT AAAATAATGA CATCTAAAAC TGACATAAGT 1200
AGCTCTGTGA TAACTTCAAT TGGAGCTATT GTATCATGCT ATGGGAAGAC AAAATGTACA 1260
GCTTCTAATA AAAATCGTGG AATCATAAAG ACTTTTCCAA TCGGGTGTGA TTATGTATCA 1320
AACAAAGGAG TTGATACTGT ATCTGTTGGT AACACACTAT ATTATGTAAA TAAGCTAGAG 1380
GGGAAAGCAC TCTATATAAA GGGTGAACCA ATTATTAATT ACTATGATCC ATTΔGTGTTT 1440
CCTTCTGATG AGTTTGATGC ATCAATTGCC CAAGTAAATG CAAAAATAAA CCAAAGCTTG 1500
GCTTTCATAC GTCGATCTGA TGAGTTACTT CATAGTGTAG ATGTAGGAAA ATCCACCACA 1560
AATGTAGTAA TTACTACTAT TATCATAGTG ATAGTTGTAG TGATATTAAT GTTAATAGCT 1620
GTAGGATTAC TGTTTTACTG TAAGACCAGG AGTACTCCTA TCATGTTAGG GAAGGATCAG 1680
CTTAGTGGTA TCAACAATCT TTCCTTTAGT AAATGAAATG CATAATGTTT ACAATCTAAA 1740
CCTCAGAATC ATAAATGTGA TGAGCTAAAT TTACTAATAC ATTCAAAAGT TCTATCCTCC 1800
AAGACCTGCA TTTTTATCAG GTCTTACATA AGCTAACCTT ACATGCTACA CTCAGCTCCA 1860
TGTTGATAGT TATATAAAA 1879
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 572 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bovine respiratory syncytial virus (B) STRAIN: FS-l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Met Gly Thr Thr Ala Met Arg Met Val He Ser He He Phe He Ser 1 5 10 15
Thr Tyr Val Thr His He Thr Leu Cys Gin Asn He Thr Glu Glu Phe 20 25 30
Tyr Gin Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu 35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr He Glu Leu Ser Lys He 50 55 60
Gin Lys Asn Val Cys Lys Ser Thr Asp Ser Lys Val Lys Leu He Lys 65 70 75 80
Gin Glu Leu Glu Arg Tyr Asn Asn Ala Val He Glu Leu Gin Ser Leu 85 90 95
Met Gin Asn Glu Pro Ala Ser Phe Ser Arg Ala Lys Arg Gly He Pro 100 105 110
Glu Leu He His Tyr Pro Arg Asn Ser Thr Lys Arg Phe Tyr Gly Leu 115 120 125
Met Gly Lys Lys Arg Lys Arg Arg His Phe Arg Phe Leu Leu Gly He 130 135 140
Gly Ser Ala He Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu 145 150 155 160
Glu Gly Glu Val Asn Lys He Lys Asn Ala Leu Leu Ser Thr Asn Lys 165 170 175
Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val 180 185 190
Leu Asp Leu Lys Asn Tyr He Asp Lys Glu Leu Leu Pro Lys Val Asn 195 200 205
Asn His Asp Cys Arg He Ser Asn He Gly Thr Val He Glu Phe Gin 210 215 220
Gin Lys Asn Asn Arg Leu Leu Glu He Ala Arg Glu Phe Ser Val Asn 225 230 235 240
Ala Gly He Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255
Leu Leu Ser Leu He Asn Asp Met Pro He Thr Asn Asp Gin Lys Lys 260 265 270 Leu Met Ser Val Cys Gin He Val Arg Gin Gin Ser Tyr Ser He Met 275 280 285
Ser Val Ser Arg Glu Val He Ala Tyr Glu Val Gin Leu Pro He Tyr 290 295 300
Gly Val He Asp Thr Pro Cys Trp Lys He His Thr Ser Pro Leu Cys 305 310 315 320
Thr Thr Asp Asn Lys Glu Gly Ser Asn He Cys Leu Thr Arg Thr Asp 325 330 335
Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gin 340 345 350
Ala Glu Thr Cys Lys Val Gin Ser Asn Arg Val Phe Cys Asp Thr Met 355 360 365
Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp He 370 375 380
Phe Asn Thr Lys Tyr Asp Cys Lys He Met Thr Ser Lys Thr Asp He 385 390 395 400
Ser Ser Ser Val He Thr Ser He Gly Ala He Val Ser Cys Tyr Gly 405 410 415
Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly He He Lys Thr 420 425 430
Phe Pro He Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val 435 440 445
Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala 450 455 460
Leu Tyr He Lys Gly Glu Pro He He Asn Tyr Tyr Asp Pro Leu Val 465 470 475 480
Phe Pro Ser Asp Glu Phe Asp Ala Ser He Ala Gin Val Asn Ala Lys 485 490 495
He Asn Gin Ser Leu Ala Phe He Arg Arg Ser Asp Glu Leu Leu His 500 505 510
Ser Val Asp Val Gly Lys Ser Thr Thr Asn Val Val He Thr Thr He 515 520 525
He He Val He Val Val Val He Leu Met Leu He Ala Val Gly Leu 530 535 540
Leu Phe Tyr Cys Lys Thr Arg Ser Thr Pro He Met Leu Gly Lys Asp 545 550 555 560
Gin Leu Ser Gly He Asn Asn Leu Ser Phe Ser Lys 565 570

Claims

WHAT IS CLAIMED IS:
1. An isolated nucleotide sequence which encodes for a protein of bovine respiratory syncytial virus (BRSV) or a fragment of said nucleotide sequence comprising 15 or more bases wherein said sequence is selected from the group consisting of the nucleocapsid protein gene, the matrix protein gene, the phosphoprotein gene, the small hydrophobic protein gene, the fusion protein gene, the glycoprotein gene and the M2 protein gene.
2. The isolated nucleotide sequence of claim 1 wherein said sequence is a genomic sequence.
3. The isolated nucleotide sequence of claim 2 wherein said sequence is isolated from BRSV strain A51908.
4. The isolated nucleotide sequence of claim 2 wherein said sequence is isolated from BRSV strain FS-l.
5. The isolated nucleotide sequence of claim 2 wherein said sequence is the nucleocapsid protein gene.
6. The isolated nucleotide sequence of claim 5 wherein said sequence is defined in the Sequence Listing as SEQ ID NO:2 or a sequence substantially homologous thereto.
7. The isolated nucleotide sequence of claim 2 wherein said sequence is the matrix protein gene.
8. The isolated nucleotide sequence of claim 7 wherein said sequence is defined in the Sequence Listing as SEQ ID NO:4 or a sequence substantially homologous thereto.
9. The isolated nucleotide sequence of claim 2 wherein said sequence is the phosphoprotein gene.
10. The isolated nucleotide sequence of claim 9 wherein said sequence is defined in the Sequence Listing as SEQ ID NO:6 or a sequence substantially homologous thereto.
11. The isolated nucleotide sequence of claim 9 wherein said sequence is defined in the Sequence Listing as SEQ ID NO:16 or a sequence substantially homologous thereto.
12. The isolated nucleotide sequence of claim 2 wherein said sequence is the small hydrophobic protein gene.
13. The isolated nucleotide sequence of claim 12 wherein said sequence is defined in the Sequence Listing as SEQ ID NO:8 or a sequence substantially homologous thereto.
14. The isolated nucleotide sequence of claim 2 wherein said sequence is the fusion protein gene.
15. The isolated nucleotide sequence of claim 14 wherein said sequence is defined in the Sequence Listing as SEQ ID NO:10 or a sequence substantially homologous thereto.
16. The isolated nucleotide sequence of claim 14 wherein said sequence is defined in the Sequence Listing as SEQ ID NO:18 or a sequence substantially homologous thereto.
17. The isolated nucleotide sequence of claim 2 wherein said sequence is the glycoprotein gene.
18. The isolated nucleotide sequence of claim 17 wherein said sequence is defined in the Sequence Listing as SEQ ID NO:12 or a sequence substantially homologous thereto.
19. The isolated nucleotide sequence of claim 2 wherein said sequence is the M2 protein gene.
20. The isolated nucleotide sequence of claim 19 wherein said sequence is defined in the Sequence Listing as SEQ ID NO:14 or a sequence substantially homologous thereto.
21. A replicative cloning vector comprising the nucleotide sequence of claim 6, 8, 10, 11, 13, 15, 16, 18 or 20 and a replicon operative in a host cell.
22. An expression system comprising the nucleotide sequence of claim 6, 8, 10, 11, 13, 15, 16, 18 or 20 operably linked to suitable control sequences.
23. The expression system of claim 22 disposed in a vector capable of replication in suitable host cells.
24. A recombinant host' cell transformed with the expression system of claim 22.
25. A method of producing a recombinant BRSV protein selected from the group consisting of nucleocapsid protein, matrix protein, phosphoprotein, small hydrophobic protein, fusion protein, glycoprotein and M2 protein comprising culturing the cells of claim 24 under conditions effective for the production of said BRSV protein.
26. A purified BRSV nucleocapsid protein or fragment thereof wherein said protein or fragment has the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:3 or a sequence substantially homologous thereto.
27. A purified BRSV matrix protein or fragment thereof wherein said protein or fragment has the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:5 or a sequence substantially homologous thereto.
28. A purified BRSV phosphoprotein or fragment thereof wherein said protein or fragment has the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:7 or a sequence substantially homologous thereto.
29. A purified BRSV small hydrophobic protein or fragment thereof wherein said protein or fragment has the amino acid sequence set forth . in the Sequence Listing as SEQ ID NO:9 or a sequence substantially homologous thereto.
30. A purified BRSV fusion protein or fragment thereof wherein said protein or fragment has the amino acid equence set forth in the Sequence Listing as SEQ ID NO:11 or a sequence substantially homologous thereto.
31. A purified BRSV glycoprotein or fragment thereof wherein said protein or fragment has the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:13 or a sequence substantially homologous thereto.
32. A purified BRSV M2 protein or fragment thereof wherein said protein or fragment has the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:15 or a sequence substantially homologous thereto.
33. A purified BRSV phosphoprotein or fragment thereof wherein said protein or fragment has the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:17 or a sequence substantially homologous thereto.
34. A purified BRSV fusion protein or fragment thereof wherein said protein or fragment has the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:19 or a sequence substantially homologous thereto.
35. An antibody to the BRSV nucleocapsid protein of claim 26.
36. An antibody to the BRSV matrix protein of claim 27.
37. An antibody to the BRSV phosphoprotein of claim 28.
38. An antibody to the BRSV small hydrophobic protein of claim 29.
39. An antibody to the BRSV fusion protein of claim 30.
40. An antibody to the BRSV glycoprotein of claim 31.
41. An antibody to the BRSV M2 protein of claim 32.
42. An antibody to the BRSV phosphoprotein of claim 33.
43. An antibody to the BRSV fusion protein of claim 34.
44. The antibody of claim 35, 36, 37, 38, 39, 40, 41, 42 or 43 which is a monoclonal antibody.
45. A BRSV vaccine comprising the purified BRSV nucleocapsid protein or fragment thereof of claim 26.
46. A BRSV vaccine comprising the purified BRSV matrix protein or fragment thereof of claim 27.
47. A BRSV vaccine comprising the purified BRSV phosphoprotein or fragment thereof of claim 28.
48. A BRSV vaccine comprising the purified BRSV small hydrophobic protein or fragment thereof of claim 29.
49. A BRSV vaccine comprising the purified BRSV fusion protein or fragment thereof of claim 30.
50. A BRSV vaccine comprising the purified BRSV glycoprotein or fragment thereof of claim 31.
51. A BRSV vaccine comprising the purified BRSV M2 protein or fragment thereof of claim 32.
52. A BRSV vaccine comprising the purified BRSV phosphoprotein or fragment thereof of claim 33.
53. A BRSV vaccine comprising the purified BRSV fusion protein or fragment thereof of claim 34.
54. A method of detecting BRSV infection using a probe comprising the isolated nucleotide sequence of claim 6, 8, 10, 11, 13, 15, 16, 18 or 20.
55. A method of detecting antibodies to BRSV using the purified BRSV nucleocapsid protein or fragment thereof of claim 26.
56. A method of detecting antibodies to BRSV using the purified BRSV matrix protein or fragment thereof of claim 27.
57. A method of detecting antibodies to BRSV using the purified BRSV phosphoprotein or fragment thereof of claim 28.
58. A method of detecting antibodies to BRSV using the purified BRSV small hydrophobic protein or fragment thereof of claim 29.
59. A method of detecting antibodies to BRSV using the purified BRSV fusion protein or fragment thereof of claim 30.
60. A method of detecting antibodies to BRSV using the purified BRSV glycoprotein or fragment thereof of claim 31.
61. A method of detecting antibodies to BRSV using the purified BRSV M2 protein or fragment thereof of claim 32.
62. A method of detecting antibodies to BRSV using the purified BRSV phosphoprotein or fragment thereof of claim 33.
63. A method of detecting antibodies to BRSV using the purified BRSV fusion protein or fragment thereof of claim 34.
PCT/US1991/008177 1990-11-05 1991-11-04 Bovine respiratory syncytial virus genes WO1992007940A2 (en)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995003070A1 (en) * 1993-07-22 1995-02-02 Syntro Corporation Recombinant swinepox virus
WO1996040945A2 (en) * 1995-06-07 1996-12-19 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
WO1997012032A1 (en) 1995-09-27 1997-04-03 The Government Of The United States Of America, As Represented By The Department Of Health And Human Services Production of infectious respiratory syncytial virus from cloned nucleotide sequences
WO1998004710A2 (en) * 1996-07-26 1998-02-05 Akzo Nobel N.V. Live recombinant bhv/brsv vaccine
US5716821A (en) * 1994-09-30 1998-02-10 Uab Research Foundation Prevention and treatment of respiratory tract disease
US5789229A (en) * 1994-09-30 1998-08-04 Uab Research Foundation Stranded RNA virus particles
WO1999024564A1 (en) * 1997-11-10 1999-05-20 University Of Maryland PRODUCTION OF NOVEL BOVINE RESPIRATORY SYNCYTIAL VIRUSES FROM cDNAs
US6017897A (en) * 1995-06-07 2000-01-25 Pasteur Merieux Connaught Canada Nucleic acid respiratory syncytial virus vaccines
US6060280A (en) * 1990-07-24 2000-05-09 The Uab Research Foundation Nucleotide sequences encoding bovine respiratory syncytial virus immunogenic proteins
US6083925A (en) * 1995-06-07 2000-07-04 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
WO2000068392A1 (en) * 1999-05-11 2000-11-16 The Board Of Trustees Of The University Of Illinois Plant-derived antigens against respiratory syncytial virus
US6730305B1 (en) 2000-05-08 2004-05-04 The Uab Research Foundation Nucleotide sequences encoding bovine respiratory syncytial virus immunogenic proteins
WO2004073737A1 (en) * 2003-02-19 2004-09-02 Merial Limited Vaccination or immunization using a prime-boost regimen against brsv, bhv-1, bvdv, bpi-3
US6908618B2 (en) 1997-11-10 2005-06-21 University Of Maryland Production of novel bovine respiratory syncytial viruses from cDNAs

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Dissertation Abstract Internatonal B, volume 51, no. 7, January 1991, University North Carolina, US; R.A. Lerch et al.: "Molecular analysis of the genes and gene products of bovine respiratory syncytial virus", page 3254, order no. DA 9034789, see the whole abstract *
J. Gen. Virol., volume 70, no. 3, March 1989, Soc. Gen. Microbiol. (GB) F. Westenbrink et al.: "Analysis of the antibody response to bovine respiratory syncytial virus proteins in calves", pages 591-601, see page 593, line 8 - page 598, line 17 (cited in the application) *
Journal of Virology, volume 63, no. 2, February 1989, Am. Soc. Microbiol., Baltimore, US, R.A. Lerch et al.: "Characterization of bovine respiratory syncytial virus proteins and mRNAs and generation of cDNA clones to the viral mRNAs", pages 833-840, see page 837, left-hand column, line 15 - page 838, left-hand column, line 2 (cited in the application) *
Journal of Virology, volume 64, no. 11, November 1990, Am. Soc. Microbiol., (Baltimore, US) R.A. Lerch et al.: "Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus", pages 5559-5569, see page 5560, left-hand column, line 58 (L documents: cited to support the non-unity) *
Virology, volume 181, no. 1, March 1991, Academic Press (New York, US) R.A. Lerch et al.: "Nucleotide sequence analysis of the bovine respiratory syncytial virus fusion protein mRNA and expression from a recombinant vaccinia virus", pages 118-131, see page 130, acknowledgments, lines 8-10 *

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US6060280A (en) * 1990-07-24 2000-05-09 The Uab Research Foundation Nucleotide sequences encoding bovine respiratory syncytial virus immunogenic proteins
WO1995003070A1 (en) * 1993-07-22 1995-02-02 Syntro Corporation Recombinant swinepox virus
US5716821A (en) * 1994-09-30 1998-02-10 Uab Research Foundation Prevention and treatment of respiratory tract disease
US5789229A (en) * 1994-09-30 1998-08-04 Uab Research Foundation Stranded RNA virus particles
US5843913A (en) * 1995-06-07 1998-12-01 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
WO1996040945A3 (en) * 1995-06-07 1997-01-23 Connaught Lab Nucleic acid respiratory syncytial virus vaccines
US6486135B1 (en) 1995-06-07 2002-11-26 Aventis Pasteur Limited Nucleic acid respiratory syncytial virus vaccines
US5880104A (en) * 1995-06-07 1999-03-09 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
US6017897A (en) * 1995-06-07 2000-01-25 Pasteur Merieux Connaught Canada Nucleic acid respiratory syncytial virus vaccines
WO1996040945A2 (en) * 1995-06-07 1996-12-19 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
US6083925A (en) * 1995-06-07 2000-07-04 Connaught Laboratories Limited Nucleic acid respiratory syncytial virus vaccines
US6677127B1 (en) 1995-06-07 2004-01-13 Aventis Pasteur Limited Nucleic acid respiratory syncytial virus vaccines
KR100905760B1 (en) * 1995-09-27 2009-10-01 더 가번먼트 오브 더 유나이티드 스테이츠 오브 아메리카, 에즈 레프리젠티드 바이 더 디파트먼트 오브 헬쓰 앤드 휴먼 서비시즈 Method of producing infectious respiratory fusion virus from cloned nucleotide sequence
WO1997012032A1 (en) 1995-09-27 1997-04-03 The Government Of The United States Of America, As Represented By The Department Of Health And Human Services Production of infectious respiratory syncytial virus from cloned nucleotide sequences
US6790449B2 (en) 1995-09-27 2004-09-14 The United States Of America As Represented By The Department Of Health And Human Services Methods for producing self-replicating infectious RSV particles comprising recombinant RSV genomes or antigenomes and the N, P, L, and M2 proteins
US6264957B1 (en) 1995-09-27 2001-07-24 The United States Of America As Represented By The Department Of Health And Human Services Product of infectious respiratory syncytial virus from cloned nucleotide sequences
WO1998004710A3 (en) * 1996-07-26 1998-04-09 Akzo Nobel Nv Live recombinant bhv/brsv vaccine
WO1998004710A2 (en) * 1996-07-26 1998-02-05 Akzo Nobel N.V. Live recombinant bhv/brsv vaccine
WO1999024564A1 (en) * 1997-11-10 1999-05-20 University Of Maryland PRODUCTION OF NOVEL BOVINE RESPIRATORY SYNCYTIAL VIRUSES FROM cDNAs
US6908618B2 (en) 1997-11-10 2005-06-21 University Of Maryland Production of novel bovine respiratory syncytial viruses from cDNAs
WO2000068392A1 (en) * 1999-05-11 2000-11-16 The Board Of Trustees Of The University Of Illinois Plant-derived antigens against respiratory syncytial virus
US8591915B2 (en) 1999-05-11 2013-11-26 Dennis E. Buetow Plant-derived vaccines against respiratory syncytial virus
US6730305B1 (en) 2000-05-08 2004-05-04 The Uab Research Foundation Nucleotide sequences encoding bovine respiratory syncytial virus immunogenic proteins
WO2004073737A1 (en) * 2003-02-19 2004-09-02 Merial Limited Vaccination or immunization using a prime-boost regimen against brsv, bhv-1, bvdv, bpi-3

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